Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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USE OF ONE OR MORE BIOMARKERS TO DETERMINE TRAUMATIC BRAIN
INJURY (TBI) IN A SUBJECT HAVING RECEIVED A HEAD COMPUTERIZED
TOMOGRAPHY SCAN THAT IS NEGATIVE FOR A TBI
RELATED APPLICATION INFORMATION
100011 This application claims priority to U.S. Application No.
17/538,572, filed
November 30, 2021, International Application No. PCT/US2021/061215, filed
November 30,
2021, U.S. Provisional Application No. 63/284,421, filed on November 30, 2021,
and U.S.
Provisional Application No. 63/294.271, filed on December 28, 2021, the
contents of each of
which are herein incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
100021 Incorporated by reference in its entirety herein is a computer-readable
nucleotide/amino acid sequence listing submitted concurrently herewith and
identified as
follows: One 7.737 byte XML file named "40136-601-SQL-ST26.xml," created on
November 29, 2022.
TECHNICAL FIELD
100031 The present disclosure relates to methods of aiding in the diagnosis
and evaluation
of a subject that has sustained, may have sustained, or is suspected of
sustaining an injury to
the head, such as a traumatic brain injury (TBI). The methods involve
detecting levels of at
least one biomarker, such as ubiquitin carboxy-terminal hydrolase Li (UCH-L1)
glial
fibrillary acidic protein (GFAP), or a combination thereof, in one or more
samples taken from
a subject at one or more time points within 24 hours after the subject has
sustained, may have
sustained, or is suspected of sustaining an injury to the head, where the
subject has also
received, at the same time, before or after (in a clinically-relevant time
frame), a head
computerized tomography (CT) scan that is negative for a TBI, and diagnosing
the subject as
more likely than not as having a TBI if the levels of one or more biomarkers
in the samples
are higher than a reference level.
BACKGROUND
100041 More than 5 million mild traumatic brain injuries (TBIs) occur each
year in the
United States alone. Currently, there is no simple, objective, accurate
measurement available
to help in patient assessment. In fact, much of TBI evaluation and diagnosis
is based on
subjective data. Unfortunately, objective measurements such as head CT and
Glasgow Coma
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Score (GCS) are not very comprehensive or sensitive in evaluating mild TBI.
Moreover, head
CT is unrevealing for the vast majority of the time for mild TBI, is
expensive, and exposes
the patient to unnecessary radiation. Additionally, a negative head CT does
not mean the
patient has been cleared from having a concussion; rather it just means
certain interventions,
such as surgery, are not warranted. Clinicians and patients need objective,
reliable
information to accurately evaluate this condition to promote appropriate
triage and recovery.
To date, limited data have been available for the use of UCH-L1 and GFAP in
the acute care
setting to aid in patient evaluation and management.
100051 Mild TBI or concussion is much harder to objectively detect and
presents an
everyday challenge in emergency care units globally. Concussion usually causes
no gross
pathology, such as hemorrhage, and no abnormalities on conventional computed
tomography
scans of the brain, but rather rapid-onset neuronal dysfunction that resolves
in a spontaneous
manner over a few days to a few weeks. Approximately 15% of mild TBI patients
suffer
persisting cognitive dysfunction. There is an unmet need for mild TBI victims
on scene, in
emergency rooms and clinics, in the sports area and in military activity
(e.g., combat).
100061 Current algorithms for assessment of the severity of brain injury
include Glasgow
Coma Scale score and other measures. These measures may at times be adequate
for relating
acute severity but are insufficiently sensitive for subtle pathology which can
result in
persistent deficit. GCS and other measures also do not enable differentiation
among types of
injury and may not be adequate. Thus, patients grouped into a single GCS level
entering a
clinical trial may have vastly heterogeneous severity and type of injury.
Because outcomes
also vary accordingly, inappropriate classification undermines the integrity
of a clinical trial.
Improved classification of injury will enable more precise delineation of
disease severity and
type for TBI patients in clinical trials.
100071 Additionally, current brain injury trials rely on outcome measures such
as Glasgow
Outcome Scale Extended, which capture global phenomena but fail to assess for
subtle
differences in outcome. Thus 30 consecutive trials for brain injury
therapeutics have failed.
Sensitive outcome measures are needed to determine how well patients have
recovered from
brain injury in order to test therapeutics and prophylactics.
SUMMARY
100081 In one aspect, the present disclosure is directed to an improvement of
a method for
aiding in the diagnosis and evaluation of a subject, such as a human subject,
that has
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sustained, may have sustained, or is suspected of sustaining an injury to the
head. Such
improved method comprises performing, simultaneously or sequentially: (1) an
assay on a
sample obtained from the subject within about 24 hours after an actual or
suspected injury to
the head to measure or detect a level of a biomarker in the sample, said
biomarker comprising
ubiquitin carboxy-terminal hydrolase Li (UCH-L1), glial fibrillary acidic
protein (GFAP), or
a combination thereof; and (2) a head computerized tomography (CT) scan on the
subject,
within a clinically-relevant time frame, wherein the improvement comprises
diagnosing the
subject as more likely than not as having traumatic brain injury (TBI) if the
level of the
biomarker is higher than a reference level and the head CT scan is negative
for a TBI.
100091 In yet another aspect, the reference level is correlated with a cutoff
level associated
with: (a) levels in subjects that have sustained a head injury; (b) the
occurrence of TBI in a
subject; (c) stage of TBI in a subject such as mild, moderate, severe, or
moderate to severe;
(d) loss of consciousness in a subject; (e) MRI positive for TBI rather than
negative; (f) the
occurrence of amnesia in a subject (i.e., amnesia present vs. absent) or (g)
severity of TBI in
a subject.
100101 In another aspect, the above-described improved method further
comprises
monitoring the subject for a TBI if the level of the biomarker is higher than
a reference level
and the head CT scan is negative for a TBI. In another aspect, the improved
method further
comprises treating the subject for a TBI if the level of the biomarker is
higher than a
reference level and the head CT scan is negative for a In yet another
aspect, the
improved method further comprises treating the subject for a TBI if the level
of the
biomarker is higher than a reference level and the head CT scan is negative
for a TBI
followed by monitoring said subject.
100111 In yet another aspect of the above-described improved method, the
sample can be
taken within about 0 to about 12 hours after an actual or suspected injury to
the head. For
example, the sample can be taken within about 5 minutes after an actual
suspected injury to
the head. Alternatively, the sample can be taken within about 10 minutes of an
actual or
suspected injury to the head. Alternatively, the sample can be taken within
about 12 minutes
of an actual or suspected injury to the head. Alternatively, the sample can be
taken within 15
minutes of an actual or suspected injury to the head. Alternatively, the
sample can be taken
within about 20 minutes of an actual or suspected injury to the head.
Alternatively, the
sample can be taken within 30 minutes of an actual or suspected injury to the
head.
Alternatively, the sample can be taken within 60 minutes of an actual or
suspected injury to
the head. Alternatively, the sample can be taken within 1.5 hours of an actual
or suspected
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injury to the head. Alternatively, the sample can be taken within 2 hours of
an actual or
suspected injury to the head. Alternatively, the sample can be taken within 3
hours of an
actual or suspected injury to the head. Alternatively, the sample can be taken
within 4 hours
of an actual or suspected injury to the head. Alternatively, the sample can be
taken within 5
hours of an actual or suspected injury to the head. Alternatively, the sample
can be taken
within 6 hours of an actual or suspected injury to the head. Alternatively,
the sample can be
taken within 7 hours of an actual or suspected injury to the head.
Alternatively, the sample
can be taken within 8 hours of an actual or suspected injury to the head.
Alternatively, the
sample can be taken within 9 hours of an actual or suspected injury to the
head.
Alternatively, the sample can be taken within 10 hours of an actual or
suspected injury to the
head. Alternatively, the sample can be taken within 11 hours of an actual or
suspected injury
to the head. Alternatively, the sample can be taken within 12 hours of an
actual or suspected
injury to the head.
100121 In one embodiment, using the above-described improved method, the
subject is
assessed or evaluated as having a TBI. In another embodiment, using the above-
described
improved method, the subject is assessed or evaluated as having a mild TBI. In
another
embodiment, using the above-described improved method, the subject is assessed
or
evaluated as having a moderate TBI. In another embodiment, using the above-
described
improved method, the subject is assessed or evaluated as having a severe TBI.
In another
embodiment, using the above-described improved method, the subject is assessed
or
evaluated as having a moderate to severe TBI. In yet still a further
embodiment, using the
above-described improved method, the subject is assessed or evaluated as not
having a TBI.
100131 The above-described improved method can further comprise treating a
subject, such
as a human subject, assessed or evaluated as having a mild, moderate, severe,
or a moderate
to severe TBI with a treatment for TBI (e.g., a surgical treatment, a
therapeutic treatment, or
combinations thereof). Any such treatment known in the art and described
further herein can
be used. Moreover, in a further embodiment, any subject being treated for TBI
can also,
optionally, be monitored during and after any course of treatment.
Alternatively, said
methods can further comprise monitoring a subject assessed as having a mild,
moderate,
severe, or a moderate to severe TBI (such as those, who yet, may not be
receiving any
treatment).
[0014] In the above-described improved method, the sample can be selected from
the group
consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a
tissue sample, a
bodily fluid sample, a saliva sample, an oropharyngeal specimen, and a
nasopharyngeal
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specimen. A blood sample can include a whole blood sample, a serum sample, or
a plasma
sample. In some embodiments, the sample is a whole blood sample. In some
embodiments,
the sample is a plasma sample. In yet other embodiments, the sample is a serum
sample. In
yet other embodiments, the sample is a saliva sample. In still yet other
embodiments, the
sample is an oropharyngeal specimen. In still other embodiments, the sample is
a
nasopharyngeal specimen. Such a sample can be obtained in a variety of ways.
For
example, the sample can be obtained after the subject sustained a head injury
caused by
physical shaking, blunt impact by an external mechanical or other force that
results in a
closed or open head trauma, one or more falls, explosions or blasts or other
types of blunt
force trauma. Alternatively, the sample can be obtained after the subject has
ingested or been
exposed to a fire, chemical, toxin or combination of a chemical and toxin.
Examples of
chemicals or toxins are mold, asbestos, a pesticide, an insecticide, an
organic solvent, a paint,
a glue, a gas, an organic metal, a drug of abuse or one or more combinations
thereof. Still
further, the sample can be obtained from a subject that suffers from an
autoimmune disease, a
metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-
2), a fungal
infection, a bacterial infection, meningitis, hydrocephalus, or any
combinations thereof.
100151 The above-described improved method can be carried out on any subject,
such as a
human subject, without regard to factors selected from the group consisting of
the subject's
clinical condition, the subject's laboratory values, the subject's
classification as suffering
from mild, moderate, severe, or a moderate to severe TBI, the subject's
exhibition of low,
moderate, or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP, and the
timing of
any event wherein the subject has sustained or may have sustained a head
injury.
100161 In the above-described improved method, the assay is an immunoassay. In
some
embodiments, the assay is a point-of-care assay. In yet other embodiments, the
assay is a
clinical chemistry assay. In yet other embodiments, the assay is a single
molecule detection
assay. In yet other embodiments, the assay is an immunoassay, the subject is a
human and
the sample is whole blood. In yet other embodiments, the assay is a point-of-
care assay, the
subject is a human and the sample is whole blood. In yet other embodiments,
the assay is a
clinical chemistry assay and the sample is whole blood. In still further
embodiments, the
assay is a single molecule detection assay and the sample is whole blood. In
yet other
embodiments, the assay is an immunoassay, the subject is a human and the
sample is serum.
In yet other embodiments, the assay is a point-of-care assay, the subject is a
human and the
sample is serum. In yet other embodiments, the assay is a clinical chemistry
assay and the
sample is serum. In still further embodiments, the assay is a single molecule
detection assay
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and the sample is serum. In yet other embodiments, the assay is an
immunoassay, the subject
is a human and the sample is plasma. In yet other embodiments, the assay is a
point-of-care
assay, the subject is a human and the sample is plasma. In yet other
embodiments, the assay
is a clinical chemistry assay and the sample is plasma. In still further
embodiments, the assay
is a single molecule detection assay and the sample is plasma. In yet other
embodiments, the
assay is an immunoassay, the subject is a human and the sample is saliva. In
yet other
embodiments, the assay is a point-of-care assay, the subject is a human and
the sample is
saliva. In yet other embodiments, the assay is a clinical chemistry assay and
the sample is
saliva. In still further embodiments, the assay is a single molecule detection
assay and the
sample is saliva. In yet other embodiments, the assay is an immunoassay, the
subject is a
human and the sample is an oropharyngeal specimen or a nasopharyngeal
specimen. In yet
other embodiments, the assay is a point-of-care assay, the subject is a human
and the sample
is an oropharyngeal specimen or a nasopharyngeal specimen. In yet other
embodiments, the
assay is a clinical chemistry assay and the sample is an oropharyngeal
specimen or a
nasopharyngeal specimen. In still further embodiments, the assay is a single
molecule
detection assay and the sample is an oropharyngeal specimen or a
nasopharyngeal specimen.
100171 In another aspect, the present disclosure is directed to a method for
aiding in the
diagnosis and evaluation of a subject, such as a human subject, that has
sustained, may have
sustained, or is suspected of sustaining an injury to the head. The method
comprises:
10018] a. performing, simultaneously or sequentially: (1) an assay on a sample
obtained
from the subject within about 24 hours after an actual or suspected injury to
the head to
measure or detect a level of a biomarker in the sample, said biomarker
comprising ubiquitin
carboxy-terminal hydrolase Li (UCH-L1), glial fibrillary acidic protein
(GFAP), or a
combination thereof; and (2) a head computerized tomography (CT) scan on the
subject
within a clinically-relevant time frame; and
10019] b. diagnosing the subject as more likely than not as having traumatic
brain injury
(TBI) if the level of the biomarker is higher than a reference level and the
head CT scan is
negative for a TBI.
100201 In yet another aspect, the reference level is correlated with a cutoff
level associated
with: (a) levels in subjects that have sustained a head injury; (b) the
occurrence of TBI in a
subject; (c) stage of TBI in a subject such as mild, moderate, severe, or
moderate to severe;
(d) loss of consciousness in a subject; (e) MRI positive for TBI rather than
negative; (f) the
occurrence of amnesia in a subject (i.e., amnesia present vs. absent) or (g)
severity of TBI in
a subject.
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[0021] In another aspect, the above-described method further comprises
monitoring the
subject for a TBI if the level of the biomarker is higher than a reference
level and the head
CT scan is negative for a TBI. In another aspect, the method further comprises
treating the
subject for a TBI if the level of the biomarker is higher than a reference
level and the head
CT scan is negative for a TBI. In yet another aspect, the method further
comprises treating
the subject for a TBI if the level of the biomarker is higher than a reference
level and the head
CT scan is negative for a TBI followed by monitoring said subject.
[0022] In yet another aspect of the above-described method, the sample can be
taken within
about 0 to about 12 hours after an actual or suspected injury to the head. For
example, the
sample can be taken within about 5 minutes after an actual suspected injury to
the head.
Alternatively, the sample can be taken within about 10 minutes of an actual or
suspected
injury to the head. Alternatively, the sample can be taken within about 12
minutes of an
actual or suspected injury to the head. Alternatively, the sample can be taken
within 15
minutes of an actual or suspected injury to the head. Alternatively, the
sample can be taken
within about 20 minutes of an actual or suspected injury to the head.
Alternatively, the
sample can be taken within 30 minutes of an actual or suspected injury to the
head.
Alternatively, the sample can be taken within 60 minutes of an actual or
suspected injury to
the head. Alternatively, the sample can be taken within 1.5 hours of an actual
or suspected
injury to the head. Alternatively, the sample can be taken within 2 hours of
an actual or
suspected injury to the head. Alternatively, the sample can be taken within 3
hours of an
actual or suspected injury to the head. Alternatively, the sample can be taken
within 4 hours
of an actual or suspected injury to the head. Alternatively, the sample can be
taken within 5
hours of an actual or suspected injury to the head. Alternatively, the sample
can be taken
within 6 hours of an actual or suspected injury to the head. Alternatively,
the sample can be
taken within 7 hours of an actual or suspected injury to the head.
Alternatively, the sample
can be taken within 8 hours of an actual or suspected injury to the head.
Alternatively, the
sample can be taken within 9 hours of an actual or suspected injury to the
head.
Alternatively, the sample can be taken within 10 hours of an actual or
suspected injury to the
head. Alternatively, the sample can be taken within 11 hours of an actual or
suspected injury
to the head. Alternatively, the sample can be taken within 12 hours of an
actual or suspected
injury to the head.
[0023] In one embodiment, using the above-described method, the subject is
assessed or
evaluated as having a TBI. In another embodiment, using the above-described
method, the
subject is assessed or evaluated as having a mild TBI. In another embodiment,
using the
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above-described method, the subject is assessed or evaluated as having a
moderate TBI. In
another embodiment, using the above-described method, the subject is assessed
or evaluated
as having a severe TB!. In another embodiment, using the above-described
method, the
subject is assessed or evaluated as having a moderate to severe TBI. In yet
still a further
embodiment, using the above-described method, the subject is assessed or
evaluated as not
having a TBI.
100241 The above-described method can further comprise treating a subject,
such as a
human subject, assessed or evaluated as having a mild, moderate, severe, or a
moderate to
severe TBI with a treatment for TBI (e.g., a surgical treatment, a therapeutic
treatment, or
combinations thereof). Any such treatment known in the art and described
further herein can
be used. Moreover, in a further embodiment, any subject being treated for TBI
can also,
optionally, be monitored during and after any course of treatment.
Alternatively, said
methods can further comprise monitoring a subject assessed as having a mild,
moderate,
severe, or a moderate to severe TBI (such as those, who as of yet, may not be
receiving any
treatment).
100251 In the above-described method, the sample can be selected from the
group
consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a
tissue sample, a
bodily fluid sample, a saliva sample, an oropharyngeal specimen, and a
nasopharyngeal
specimen. In some embodiments, the sample is a whole blood sample. A blood
sample can
be a whole blood sample, a serum sample, or a plasma sample. In some
embodiments, the
sample is a plasma sample. In yet other embodiments, the sample is a serum
sample. In yet
other embodiments, the sample is a saliva sample. In still yet other
embodiments, the sample
is an oropharyngeal specimen. In still other embodiments, the sample is a
nasopharyngeal
specimen. Such a sample can be obtained in a variety of ways. For example, the
sample can
be obtained after the subject sustained a head injury caused by physical
shaking, blunt impact
by an external mechanical or other force that results in a closed or open head
trauma, one or
more falls, explosions or blasts or other types of blunt force trauma.
Alternatively, the
sample can be obtained after the subject has ingested or been exposed to a
fire, chemical,
toxin or combination of a chemical and toxin. Examples of chemicals or toxins
are mold,
asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a
gas, an organic
metal, a drug of abuse or one or more combinations thereof. Still further, the
sample can be
obtained from a subject that suffers from an autoimmune disease, a metabolic
disorder, a
brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal
infection, a bacterial
infection, meningitis, hydrocephalus, or any combinations thereof.
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100261 The above-described method can be carried out on any subject, such as a
human
subject, without regard to factors selected from the group consisting of the
subject's clinical
condition, the subject's laboratory values, the subject's classification as
suffering from mild,
moderate, severe, or a moderate to severe TBI, the subject's exhibition of
low, moderate, or
high levels of UCH-L1, GFAP and or UCH-L1 and GFAP, and the timing of any
event
wherein the subject has sustained or may have sustained a head injury.
100271 In the above-described method, the assay is an immunoassay. In some
embodiments, the assay is a point-of-care assay. In yet other embodiments, the
assay is a
clinical chemistry assay. In yet other embodiments, the assay is a single
molecule detection
assay. In yet other embodiments, the assay is an immunoassay, the subject is a
human and
the sample is whole blood. In yet other embodiments, the assay is a point-of-
care assay, the
subject is a human and the sample is whole blood. In yet other embodiments,
the assay is a
clinical chemistry assay and the sample is whole blood. In still further
embodiments, the
assay is a single molecule detection assay and the sample is whole blood. In
yet other
embodiments, the assay is an immunoassay, the subject is a human and the
sample is serum.
In yet other embodiments, the assay is a point-of-care assay, the subject is a
human and the
sample is serum. In yet other embodiments, the assay is a clinical chemistry
assay and the
sample is serum. In still further embodiments, the assay is a single molecule
detection assay
and the sample is serum. In yet other embodiments, the assay is an
immunoassay, the subject
is a human and the sample is plasma. In yet other embodiments, the assay is a
point-of-care
assay, the subject is a human and the sample is plasma. In yet other
embodiments, the assay
is a clinical chemistry assay and the sample is plasma. In still further
embodiments, the assay
is a single molecule detection assay and the sample is plasma. In yet other
embodiments, the
assay is an immunoassay, the subject is a human and the sample is saliva. In
yet other
embodiments, the assay is a point-of-care assay, the subject is a human and
the sample is
saliva. In yet other embodiments, the assay is a clinical chemistry assay and
the sample is
saliva. In still further embodiments, the assay is a single molecule detection
assay and the
sample is saliva. In yet other embodiments, the assay is an immunoassay, the
subject is a
human and the sample is an oropharyngeal specimen or a nasopharyngeal
specimen. In yet
other embodiments, the assay is a point-of-care assay, the subject is a human
and the sample
is an oropharyngeal specimen or a nasopharyngeal specimen. In yet other
embodiments, the
assay is a clinical chemistry assay and the sample is an oropharyngeal
specimen or a
nasopharyngeal specimen. In still further embodiments, the assay is a single
molecule
detection assay, and the sample is an oropharyngeal specimen or a
nasopharyngeal specimen.
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[0028] In still yet another aspect, the present disclosure relates to an
improvement of a
method for aiding in the diagnosis and evaluation of a subject, such as a
human subject, that
has sustained or may have sustained an injury to the head. The improvement
comprising
performing an assay on a sample obtained from the subject within about 24
hours after an
actual or suspected injury to the head to measure or detect a level of a
biomarker in the
sample, said biomarker comprising ubiquitin carboxy-terminal hydrolase Li (UCH-
L1), glial
fibrillary acidic protein (GFAP), or a combination thereof; and wherein the
improvement
comprises diagnosing the subject as more likely than not as having traumatic
brain injury
(TBI) if the level of the biomarker is higher than a reference level, and
either a head
computerized tomography (CT) scan on the subject within a clinically-relevant
time frame is
negative for a TBI, or no head CT scan is performed on the subject.
[0029] In yet another aspect, the reference level is correlated with a cutoff
level associated
with: (a) levels in subjects that have sustained a head injury; (b) the
occurrence of TBI in a
subject; (c) stage of TBI in a subject such as mild, moderate, severe, or
moderate to severe;
(d) loss of consciousness in a subject; (e) MRI positive for TBI rather than
negative; (f) the
occurrence of amnesia in a subject (i.e., amnesia present vs. absent) or (g)
severity of TBI in
a subject.
[0030] In another aspect, the above-described improved method further
comprises
monitoring the subject for a TBI if the level of the biomarker is higher than
a reference level
and optionally, if performed, a head Cl scan is negative for a TBI. In another
aspect, the
improved method further comprises treating the subject for a TBI if the level
of the
biomarker is higher than a reference level and, optionally, if performed, if
the head CT scan
is negative for a TBI. In yet another aspect, the improved method further
comprises treating
the subject for a TBI if the level of the biomarker is higher than a reference
level and
optionally, if performed, if the head CT scan is negative for a TBI followed
by monitoring
said subject.
[0031] In yet another aspect of the above-described improved method, the
sample can be
taken within about 0 to about 12 hours after an actual or suspected injury to
the head. For
example, the sample can be taken within about 5 minutes after an actual
suspected injury to
the head. Alternatively, the sample can be taken within about 10 minutes of an
actual or
suspected injury to the head. Alternatively, the sample can be taken within
about 12 minutes
of an actual or suspected injury to the head. Alternatively, the sample can be
taken within 15
minutes of an actual or suspected injury to the head. Alternatively, the
sample can be taken
within about 20 minutes of an actual or suspected injury to the head.
Alternatively, the
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sample can be taken within 30 minutes of an actual orsuspected injury to the
head.
Alternatively, the sample can be taken within 60 minutes of an actual or
suspected injury to
the head. Alternatively, the sample can be taken within 1.5 hours of an actual
or suspected
injury to the head. Alternatively, the sample can be taken within 2 hours of
an actual or
suspected injury to the head. Alternatively, the sample can be taken within 3
hours of an
actual or suspected injury to the head. Alternatively, the sample can be taken
within 4 hours
of an actual or suspected injury to the head. Alternatively, the sample can be
taken within 5
hours of an actual or suspected injury to the head. Alternatively, the sample
can be taken
within 6 hours of an actual or suspected injury to the head. Alternatively,
the sample can be
taken within 7 hours of an actual or suspected injury to the head.
Alternatively, the sample
can be taken within 8 hours of an actual or suspected injury to the head.
Alternatively, the
sample can be taken within 9 hours of an actual or suspected injury to the
head.
Alternatively, the sample can be taken within 10 hours of an actual or
suspected injury to the
head. Alternatively, the sample can be taken within 11 hours of an actual or
suspected injury
to the head. Alternatively, the sample can be taken within 12 hours of an
actual or suspected
injury to the head.
100321 In one embodiment, using the above-described improved method, the
subject is
assessed or evaluated as having a TBI. In another embodiment, using the above-
described
improved method, the subject is assessed or evaluated as having a mild TBI. In
another
embodiment, using the above-described improved method, the subject is assessed
or
evaluated as having a moderate TBI. In another embodiment, using the above-
described
improved method, the subject is assessed or evaluated as having a severe TBI.
In another
embodiment, using the above-described improved method, the subject is assessed
or
evaluated as having a moderate to severe TBI. In yet still a further
embodiment, using the
above-described improved method, the subject is assessed or evaluated as not
having a TBI.
10033] The above-described improved method can further comprise treating a
subject, such
as a human subject, assessed or evaluated as having a mild, moderate, severe,
or a moderate
to severe TBI with a treatment for TBI (e.g., a surgical treatment, a
therapeutic treatment, or
combinations thereof). Any such treatment known in the art and described
further herein can
be used. Moreover, in a further embodiment, any subject being treated for TBI
can also,
optionally, be monitored during and after any course of treatment.
Alternatively, said
methods can further comprise monitoring a subject assessed as having a mild,
moderate,
severe, or a moderate to severe TBI (such as those, who yet may not be
receiving any
treatment).
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100341 In the above-described improved method, the sample can be selected from
the group
consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a
tissue sample, a
bodily fluid sample, a saliva sample, an oropharyngeal specimen, and a
nasopharyngeal
specimen. A blood sample can be a whole blood sample, a serum sample, or a
plasma sample.
In some embodiments, the sample is a whole blood sample. In some embodiments,
the
sample is a plasma sample. In yet other embodiments, the sample is a serum
sample. In yet
other embodiments, the sample is a saliva sample. In still yet other
embodiments, the sample
is an oropharyngeal specimen. In still other embodiments, the sample is a
nasopharyngeal
specimen. Such a sample can be obtained in a variety of ways. For example, the
sample can
be obtained after the subject sustained a head injury caused by physical
shaking, blunt impact
by an external mechanical or other force that results in a closed or open head
trauma, one or
more falls, explosions or blasts or other types of blunt force trauma.
Alternatively, the
sample can be obtained after the subject has ingested or been exposed to a
fire, chemical,
toxin or combination of a chemical and toxin. Examples of chemicals or toxins
are mold,
asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a
gas, an organic
metal, a drug of abuse or one or more combinations thereof. Still further, the
sample can be
obtained from a subject that suffers from an autoimmune disease, a metabolic
disorder, a
brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal
infection, a bacterial
infection, meningitis, hydrocephalus, or any combinations thereof.
10035] 'lite above-described improved method can be carried out on any
subject, such as a
human subject, without regard to factors selected from the group consisting of
the subject's
clinical condition, the subject's laboratory values, the subject's
classification as suffering
from mild, moderate, severe, or a moderate to severe TBI, the subject's
exhibition of low,
moderate or high levels of UCH-L1, GFAP and or UCH-L1 and GFAP, and the timing
of any
event wherein the subject has sustained or may have sustained a head injury.
10036] In the above-described improved method, the assay is an immunoassay. In
some
embodiments, the assay is a point-of-care assay. In yet other embodiments, the
assay is a
clinical chemistry assay. In yet other embodiments, the assay is a single
molecule detection
assay. In yet other embodiments, the assay is an immunoassay, the subject is a
human and
the sample is whole blood. In yet other embodiments, the assay is a point-of-
care assay, the
subject is a human and the sample is whole blood. In yet other embodiments,
the assay is a
clinical chemistry assay, and the sample is whole blood. In still further
embodiments, the
assay is a single molecule detection assay, and the sample is whole blood. In
yet other
embodiments, the assay is an immunoassay, the subject is a human and the
sample is serum.
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In yet other embodiments, the assay is a point-of-care assay, the subject is a
human and the
sample is serum. In yet other embodiments, the assay is a clinical chemistry
assay, and the
sample is serum. In still further embodiments, the assay is a single molecule
detection assay,
and the sample is serum. In yet other embodiments, the assay is an
immunoassay, the subject
is a human and the sample is plasma. In yet other embodiments, the assay is a
point-of-care
assay, the subject is a human and the sample is plasma. In yet other
embodiments, the assay
is a clinical chemistry assay, and the sample is plasma. In still further
embodiments, the
assay is a single molecule detection assay, and the sample is plasma. In yet
other
embodiments, the assay is an immunoassay, the subject is a human and the
sample is saliva.
In yet other embodiments, the assay is a point-of-care assay, the subject is a
human and the
sample is saliva. In yet other embodiments, the assay is a clinical chemistry
assay, and the
sample is saliva. In still further embodiments, the assay is a single molecule
detection assay,
and the sample is saliva. In yet other embodiments, the assay is an
immunoassay, the subject
is a human and the sample is an oropharyngeal specimen or a nasopharyngeal
specimen. In
yet other embodiments, the assay is a point-of-care assay, the subject is a
human and the
sample is an oropharyngeal specimen or a nasopharyngeal specimen. In yet other
embodiments, the assay is a clinical chemistry assay, and the sample is an
oropharyngeal
specimen or a nasopharyngeal specimen. In still further embodiments, the assay
is a single
molecule detection assay, and the sample is an oropharyngeal specimen or a
nasopharyngeal
specimen.
100371 In the above-described improved method, the reference level for GFAP is
from
about 30 pg/mL to about 1700 pg/mL and the reference level for UCH-L1 is from
about 150
pg/mL to about 700 pg/mL. In some embodiments, the reference level for GFAP
from about
90 pg/mL to about 1680 pg/mL and the reference level for UCH-L1 is from about
220 pg/mL
to about 670 pg/mL. In other embodiments, the reference level for GFAP is from
about 110
pg/mL to about 950 pg/mL and the reference level for UCH-L1 is from about 160
pg/mL to
about 320 pg/mL._
BRIEF DESCRIPTION OF THE DRAWINGS
100381 FIGS. 1A-D show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
Glasgow Coma Score (GCS) severity (i.e., GCS mild vs. moderate/severe) for
those subjects
having CT scans negative for TBI. Samples were assessed within 12 hours from
injury (FIG.
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1A, 1C) or within 24.1 hours from injury (FIG. 1B, 1D). GFAP levels are shown
in FIG. 1A
and FIG. 1B. UCH-L1 levels are shown in FIG. 1C and FIG. 1D.
100391 FIGS. 2A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
loss of consciousness after injury (i.e., present vs. absent) for those
subjects having CT scans
negative for TBI. Samples were assessed within 4 hours (FIG. 2A, 2D), within
12 hours
(FIG. 2B, 2E), or within 24.1 hours from injury (FIG. 2C, 2F). GFAP levels are
shown in
FIG. 2A-2C. UCH-L1 levels are shown in FIG. 2D-F.
100401 FIGS. 3A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
MRI results (i.e., positive vs. negative) for those subjects having CT scans
negative for TBI.
Samples were assessed within 4 hours (FIG. 3A, 3D), within 12 hours (FIG. 3B,
3E) or
within 24.1 hours from injury (FIG. 3C, 3F). GFAP levels are shown in FIGS. 3A-
3C.
UCH-L1 levels are shown in FIG. 3D-F.
[0041] FIGS. 4A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
post-traumatic amnesia (i.e., amnesia present vs. absent) for those subjects
having CT scans
negative for TBI. Samples were assessed within 4 hours (FIG. 4A, 4D), within
12 hours
(FIG. 4B, 4E) or within 24.1 hours from injury (FIG. 4C, 4F). GFAP levels are
shown in
FIGS. 4A-C. UCH-L1 levels are shown in FIGS. 4D-F.
DETAILED DESCRIPTION
[0042] The present disclosure relates to improved methods for aiding in the
diagnosis and
evaluation of a subject, such as a human subject, that has sustained, may have
sustained, or is
suspected of sustaining an injury to the head, such as a traumatic brain
injury (e.g., such as a
mild, moderate, severe, or moderate to severe TBI), using a biomarker, such as
ubiquitin
carboxy-terminal hydrolase Li (UCH-L1), glial fibrillary acidic protein
(GFAP), or a
combination thereof, when the subject has optionally received, at least one
head
computerized tomography (CT) scan, within a clinically-relevant time frame,
that is negative
for a TBI.
[0043] The methods described herein involve detecting one or more biomarker
levels in one
or more samples taken from the subject, such as a human subject, at a time
point within about
24 hours of an actual injury or suspected injury to the head. Optionally, if
performed, the
head CT scan can be performed either simultaneously or sequentially, in any
order, at the
same time, prior to, or after one or more biomarker levels are detected in one
or more
samples taken from the subject. The detection of levels of the one or more
biomarkers, such
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as UCH-L1, GFAP, or combination thereof, that are higher than reference levels
provide an
aid in accurately evaluating or diagnosing subjects, such as human subjects,
as more likely
than not as having a TB1 when said subjects have optionally received a head CT
scan that is
negative for TBI. In other words, in some aspects, the improved methods
described herein
allow for the identification of subjects who have suffered a TBI but may
otherwise have been
incorrectly diagnosed as not having a TBI if such a diagnosis was based solely
on the result
of the one or more head CT scans.
[0044] Section headings as used in this section and the entire disclosure
herein are merely
for organizational purposes and are not intended to be limiting.
1. Definitions
[0045] Unless otherwise defined, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art. In
case of
conflict, the present document, including definitions, will control. Preferred
methods and
materials are described below, although methods and materials similar or
equivalent to those
described herein can be used in practice or testing of the present invention.
All publications,
patent applications, patents and other references mentioned herein are
incorporated by
reference in their entirety. The materials, methods, and examples disclosed
herein are
illustrative only and not intended to be limiting.
[0046] The terms "comprise(s)," "include(s)," "having," "has," "can,"
"contain(s)," and
variants thereof, as used herein, are intended to be open-ended transitional
phrases, terms, or
words that do not preclude the possibility of additional acts or structures.
The singular forms
"a," "and" and "the" include plural references unless the context clearly
dictates otherwise.
The present disclosure also contemplates other embodiments "comprising,-
"consisting of'
and "consisting essentially of," the embodiments or elements presented herein,
whether
explicitly set forth or not.
[0047] For the recitation of numeric ranges herein, each intervening number
there between
with the same degree of precision is explicitly contemplated. For example, for
the range of 6-
9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the
range 6.0-7.0, the
number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are
explicitly contemplated.
[0048] "Affinity matured antibody" is used herein to refer to an antibody with
one or more
alterations in one or more CDRs, which result in an improvement in the
affinity (i.e., KD, kd
or ka) of the antibody for a target antigen compared to a parent antibody,
which does not
possess the alteration(s). Exemplary affinity matured antibodies will have
nanomolar or even
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picomolar affinities for the target antigen. A variety of procedures for
producing affinity
matured antibodies is known in the art, including the screening of a
combinatory antibody
library that has been prepared using bio-display. For example, Marks et al.,
BioTechnology,
10: 779-783 (1992) describes affinity maturation by VH and VL domain
shuffling. Random
mutagenesis of CDR and/or framework residues is described by Barbas et al.,
Proc. Nat.
Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155
(1995); Yelton et
al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7):
3310-3319
(1995); and Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective
mutation at
selective mutagenesis positions and at contact or hypermutation positions with
an activity-
enhancing amino acid residue is described in U.S. Patent No. 6,914,128 B 1.
[0049] An "analog assay" as used herein refers to an assay in which the
presence of and/or
concentration of an analyte in a test sample is determined by measuring the
total signal
produced (e.g., fluorescence, color, etc.) by the analyte in an entire
reaction mixture (e.g., a
single reaction vessel). In an analog assay, the noise is indistinguishable
from the signal. An
example of an analog assay is an assay in which the presence of and/or
concentration of an
analyte is determined by measuring the total signal produced from a plurality
of beads or
microparticles contained in a single reaction vessel.
[0050] "Antibody" and "antibodies" as used herein refers to monoclonal
antibodies,
multispecific antibodies, human antibodies, humanized antibodies (fully or
partially
humanized), animal antibodies such as, but not limited to, a bird (for
example, a duck or a
goose), a shark, a whale, and a mammal, including a non-primate (for example,
a cow, a pig,
a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig,
a cat, a dog, a rat,
a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee,
etc.),
recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFv"), single
chain
antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab')2
fragments,
disulfide-linked Fvs ("sdFv"), and anti-idiotypic ("anti-Id") antibodies, dual-
domain
antibodies, dual variable domain (DVD) or triple variable domain (TVD)
antibodies (dual-
variable domain immunoglobulins and methods for making them are described in
Wu, C., et
al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International
Application WO
2001/058956, the contents of each of which are herein incorporated by
reference), and
functionally active epitope-binding fragments of any of the above. Antibodies
include
immunoglobulin molecules and immunologically active fragments of
immunoglobulin
molecules, namely, molecules that contain an analyte-binding site.
Immunoglobulin
molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY),
class (for
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example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity
sake, an
antibody against an analyte is frequently referred to herein as being either
an "anti-analyte
antibody" or merely an "analyte antibody" (e.g., an anti-UCH-L1 antibody or a
UCH-L1
antibody).
[0051] "Antibody fragment" as used herein refers to a portion of an intact
antibody
comprising the antigen-binding site or variable region. The portion does not
include the
constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the
antibody isotype)
of the Fc region of the intact antibody. Examples of antibody fragments
include, but are not
limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2
fragments, Fd
fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-
chain
polypeptides containing only one light chain variable domain, single-chain
polypeptides
containing the three CDRs of the light-chain variable domain, single-chain
polypeptides
containing only one heavy chain variable region, and single-chain polypeptides
containing
the three CDRs of the heavy chain variable region.
[0052] The "area under curve" or "AUC" refers to area under a ROC curve. AUC
under a
ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test,
whereas an
AUC of 0.5 represents an insignificant test. A preferred AUC may be at least
approximately
0.700, at least approximately 0.750, at least approximately 0.800, at least
approximately
0.850, at least approximately 0.900, at least approximately 0.910, at least
approximately
0.920, at least approximately 0.930, at least approximately 0.940, at least
approximately
0.950, at least approximately 0.960, at least approximately 0.970, at least
approximately
0.980, at least approximately 0.990, or at least approximately 0.995.
100531 "Bead" and "particle" are used herein interchangeably and refer to a
substantially
spherical solid support. One example of a bead or particle is a microparticle.
Microparticles
that can be used herein can be any type known in the art. For example, the
bead or particle
can be a magnetic bead or magnetic particle. Magnetic beads/particles may be
ferromagnetic,
ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary
ferromagnetic
materials include Fe, Co, Ni, Gd, Dy, Cr02, MnAs, MnBi, Eu0, and NiO/Fe.
Examples of
ferrimagnetic materials include NiFe204, CoFe204, Fe304 (or Fe0Fe203). Beads
can have a
solid core portion that is magnetic and is surrounded by one or more non-
magnetic layers.
Alternately, the magnetic portion can be a layer around a non-magnetic core.
The
microparticles can be of any size that would work in the methods described
herein, e.g., from
about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to
about 3 nm.
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100541 "Binding protein" is used herein to refer to a monomeric or multimeric
protein that
binds to and forms a complex with a binding partner, such as, for example, a
polypeptide, an
antigen, a chemical compound or other molecule, or a substrate of any kind. A
binding
protein specifically binds a binding partner. Binding proteins include
antibodies, as well as
antigen-binding fragments thereof and other various forms and derivatives
thereof as are
known in the art and described herein below, and other molecules comprising
one or more
antigen-binding domains that bind to an antigen molecule or a particular site
(epitope) on the
antigen molecule. Accordingly, a binding protein includes, but is not limited
to, an antibody
a tetrameric immunoglobulin, an IgG molecule, an IgG1 molecule, a monoclonal
antibody, a
chimeric antibody, a CDR-grafted antibody, a humanized antibody, an affinity
matured
antibody, and fragments of any such antibodies that retain the ability to bind
to an antigen.
10055] "Bispecific antibody" is used herein to refer to a full-length antibody
that is
generated by quadroma technology (see Milstein et al., Nature, 305(5934): 537-
540 (1983)),
by chemical conjugation of two different monoclonal antibodies (see, Staerz et
al., Nature,
314(6012): 628-631 (1985)), or by knob-into-hole or similar approaches, which
introduce
mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA,
90(14): 6444-6448
(1993)), resulting in multiple different immunoglobulin species of which only
one is the
functional bispecific antibody. A bispecific antibody binds one antigen (or
epitope) on one of
its two binding arms (one pair of HC/LC), and binds a different antigen (or
epitope) on its
second arm (a different pair of HC/LC). By this definition, a bispecific
antibody has two
distinct antigen-binding arms (in both specificity and CDR sequences) and is
monovalent for
each antigen to which it binds to.
100561 "CDR" is used herein to refer to the "complementarity determining
region" within
an antibody variable sequence. There are three CDRs in each of the variable
regions of the
heavy chain and the light chain. Proceeding from the N-terminus of a heavy or
light chain,
these regions are denoted "CDR1", "CDR2", and "CDR3", for each of the variable
regions.
The term "CDR set" as used herein refers to a group of three CDRs that occur
in a single
variable region that binds the antigen. An antigen-binding site, therefore,
may include six
CDRs, comprising the CDR set from each of a heavy and a light chain variable
region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be
referred to as
a "molecular recognition unit." Crystallographic analyses of antigen-antibody
complexes
have demonstrated that the amino acid residues of CDRs form extensive contact
with bound
antigen, wherein the most extensive antigen contact is with the heavy chain
CDR3. Thus, the
molecular recognition units may be primarily responsible for the specificity
of an antigen-
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binding site. In general, the CDR residues are directly and most substantially
involved in
influencing antigen binding.
[0057] The exact boundaries of these CDRs have been defined differently
according to
different systems. The system described by Kabat (Kabat et al., Sequences of
Proteins of
Immunological Interest (National Institutes of Health, Bethesda, Md. (1987)
and (1991)) not
only provides an unambiguous residue numbering system applicable to any
variable region of
an antibody, but also provides precise residue boundaries defining the three
CDRs. These
CDRs may be referred to as "Kabat CDRs". Chothia and coworkers (Chothia and
Lesk, J.
Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883
(1989)) found that
certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone
conformations, despite having great diversity at the level of amino acid
sequence. These sub-
portions were designated as "LI", "L2", and "L3", or "Hl", "H2", and "H3",
where the "L"
and the "H" designate the light chain and the heavy chain regions,
respectively. These
regions may be referred to as "Chothia CDRs", which have boundaries that
overlap with
Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs
have been
described by Padlan, FASEB J., 9: 133-139 (1995), and MacCallum, J. Mal.
Biol., 262(5):
732-745 (1996). Still other CDR boundary definitions may not strictly follow
one of the
herein systems, but will nonetheless overlap with the Kabat CDRs, although
they may be
shortened or lengthened in light of prediction or experimental findings that
particular residues
or groups of residues or even entire CDRs do not significantly impact antigen
binding. "lhe
methods used herein may utilize CDRs defined according to any of these
systems, although
certain embodiments use Kabat- or Chothia-defined CDRs.
[0058] A "clinically-relevant time frame" refers to a time frame (e.g.,
seconds, minutes, or
hours) during which a careful and prudent medical practitioner (e.g., doctor,
nurse,
paramedic, or other) would reasonably consider the results of one or more
biomarker tests to
have bearing on an imaging procedure, such as a head CT scan, or pursuant to
guidelines
established by an overseeing entity (e.g., a standards-setting body such as
the World Health
Organization (WHO), physicians review board, regulatory approval authority
such as FDA,
EMEA or other, etc.).
[0059] "Component," "components," or "at least one component," refer generally
to a
capture antibody, a detection or conjugate a calibrator, a control, a
sensitivity panel, a
container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme,
a detection
reagent, a pretreatment reagent/solution, a substrate (e.g., as a solution), a
stop solution, and
the like that can be included in a kit for assay of a test sample, such as a
patient urine, whole
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blood, serum or plasma sample, in accordance with the methods described herein
and other
methods known in the art. Some components can be in solution or lyophilized
for
reconstitution for use in an assay.
[0060] "Correlated to" as used herein refers to compared to.
[0061] "CT scan" as used herein refers to a computerized tomography (CT) scan.
A CT
scan combines a series of X-ray images taken from different angles and uses
computer
processing to create cross-sectional images, or slices, of the bones, blood
vessels and soft
tissues inside your body. The CT scan may use X-ray CT, positron emission
tomography
(PET), single-photon emission computed tomography (SPECT), computed axial
tomography
(CAT scan), or computer aided tomography. The CT scan may be a conventional CT
scan or
a spiral/helical CT scan. In a conventional CT scan, the scan is taken slice
by slice and after
each slice the scan stops and moves down to the next slice, e.g., from the top
of the abdomen
down to the pelvis. The conventional CT scan requires patients to hold their
breath to avoid
movement artefact. The spiral/helical CT scan is a continuous scan which is
taken in a spiral
fashion and is a much quicker process where the scanned images are contiguous.
[0062] A head CT scan is "negative" for a TBI when no intracranial lesion(s)
is observed in
an image taken from a subject that has sustained, may have sustained or is
suspected of
sustaining an injury to the head. To further clarify, the head CT scan of a
subject is
"negative- for a TBI when a lesion is not found or identified; however, in
some aspects, the
subject may still be experiencing symptoms (e.g., of TBI) even though the head
Cl is
negative. Most subjects will be negative for a TBI on head CT given that not
all injuries or
lesions can be visualized by head CT. Consequently, the methods and assays
described herein
can be used to provide an assessment or determination of a subject with a
negative head CT
that may still have a TBI.
[0063] "Determined by an assay" is used herein to refer to the determination
of a reference
level by any appropriate assay. The determination of a reference level may, in
some
embodiments, be achieved by an assay of the same type as the assay that is to
be applied to
the sample from the subject (for example, by an immunoassay, clinical
chemistry assay, a
single molecule detection assay, protein immunoprecipitation,
immunoelectrophoresis,
chemical analysis, SDS-PAGE and Western blot analysis, or protein
immunostaining,
electrophoresis analysis, a protein assay, a competitive binding assay, a
functional protein
assay, or chromatography or spectrometry methods, such as high-performance
liquid
chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS)). The
determination of a reference level may, in some embodiments, be achieved by an
assay of the
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same type and under the same assay conditions as the assay that is to be
applied to the sample
from the subject. As noted herein, this disclosure provides exemplary
reference levels (e.g.,
calculated by comparing reference levels at different time points). It is well
within the
ordinary skill of one in the art to adapt the disclosure herein for other
assays to obtain assay-
specific reference levels for those other assays based on the description
provided by this
disclosure. For example, a set of training samples comprising samples obtained
from human
subjects known to have sustained an injury to the head (and more particularly,
samples
obtained from human subjects known to have sustained a (i) mild TBI; and/or
(ii) moderate,
severe, or moderate to severe TBI and samples obtained from human subjects
known not to
have sustained an injury to the head may be used to obtain assay-specific
reference levels. It
will be understood that a reference level "determined by an assay" and having
a recited level
of "sensitivity" and/or "specificity" is used herein to refer to a reference
level which has been
determined to provide a method of the recited sensitivity and/or specificity
when said
reference level is adopted in the methods of the invention. It is well within
the ordinary skill
of one in the art to determine the sensitivity and specificity associated with
a given reference
level in the methods of the invention, for example by repeated statistical
analysis of assay
data using a plurality of different possible reference levels.
[0064] Practically, when discriminating between a subject as having a
traumatic brain
injury or not having a traumatic brain injury or a subject as having a mild
versus a moderate,
severe, or moderate to severe traumatic brain injury, the skilled person will
balance the effect
of raising a cutoff on sensitivity and specificity. Raising or lowering a
cutoff will have a well-
defined and predictable impact on sensitivity and specificity, and other
standard statistical
measures. It is well known that raising a cutoff will improve specificity but
is likely to
worsen sensitivity (proportion of those with disease who test positive). In
contrast, lowering
a cutoff will improve sensitivity but will worsen specificity (proportion of
those without
disease who test negative). The ramifications for detecting traumatic brain
injury or
determining a mild versus moderate, severe, or moderate to severe traumatic
brain injury will
be readily apparent to those skilled in the art. In discriminating whether a
subject has or does
not have a traumatic brain injury or a mild versus a moderate, severe, or
moderate to severe
traumatic brain injury, the higher the cutoff, specificity improves as more
true negatives (i.e.,
subjects not having a traumatic brain injury, not having a mild traumatic
brain injury, not
have a moderate traumatic brain injury, not having a severe traumatic brain
injury or not
having a moderate to severe traumatic brain injury) are distinguished from
those having a
traumatic brain injury, a mild traumatic brain injury, a moderate traumatic
brain injury, a
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severe traumatic brain injury or a moderate to severe traumatic brain injury.
But at the same
time, raising the cutoff decreases the number of cases identified as positive
overall, as well as
the number of true positives, so the sensitivity must decrease. Conversely,
the lower the
cutoff, sensitivity improves as more true positives (i.e., subjects having a
traumatic brain
injury, having a mild traumatic brain injury, having a moderate traumatic
brain injury, having
a severe traumatic brain injury or having a moderate to severe traumatic brain
injury) are
distinguished from those who do not have a traumatic brain injury, a mild
traumatic brain
injury, a moderate traumatic brain injury, a severe traumatic brain injury or
a moderate to
severe traumatic brain injury. But at the same time, lowering the cutoff
increases the number
of cases identified as positive overall, as well as the number of false
positives, so the
specificity must decrease.
[0065] Generally, a high sensitivity value helps one of skill rule out disease
or condition
(such as a traumatic brain injury, mild traumatic brain injury, moderate
traumatic brain
injury, severe traumatic brain injury or moderate to severe traumatic brain
injury), and a high
specificity value helps one of skill rule in disease or condition. Whether one
of skill desires
to rule out or rule in disease depends on what the consequences are for the
patient for each
type of error. Accordingly, one cannot know or predict the precise balancing
employed to
derive a test cutoff without full disclosure of the underlying information on
how the value
was selected. The balancing of sensitivity against specificity and other
factors will differ on a
case-by-case basis. This is why it is sometimes preferable to provide
alternate cutoff (e.g.,
reference) values so a physician or practitioner can choose.
100661 "Derivative" of an antibody as used herein may refer to an antibody
having one or
more modifications to its amino acid sequence when compared to a genuine or
parent
antibody and exhibit a modified domain structure. The derivative may still be
able to adopt
the typical domain configuration found in native antibodies, as well as an
amino acid
sequence, which is able to bind to targets (antigens) with specificity.
Typical examples of
antibody derivatives are antibodies coupled to other polypeptides, rearranged
antibody
domains, or fragments of antibodies. The derivative may also comprise at least
one further
compound, e.g., a protein domain, said protein domain being linked by covalent
or non-
covalent bonds. The linkage can be based on genetic fusion according to the
methods known
in the art. The additional domain present in the fusion protein comprising the
antibody may
preferably be linked by a flexible linker, advantageously a peptide linker,
wherein said
peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a
length
sufficient to span the distance between the C-terminal end of the further
protein domain and
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the N-terminal end of the antibody or vice versa. The antibody may be linked
to an effector
molecule having a conformation suitable for biological activity or selective
binding to a solid
support, a biologically active substance (e.g., a cytokine or growth hormone),
a chemical
agent, a peptide, a protein, or a drug, for example.
10067] "Digital assay" as used herein refers to an assay in which an analyte
is captured and
a molecule of the analyte segregated and interrogated (e.g., to detect the
presence and/or
concentration of the analyte in a sample). In a digital assay, noise is
separated from signal.
In a digital assay, the results are assigned a value of 1 or 0. Examples of
digital assays
include one or more of the following (which may overlap but are not mutually
exclusive):
single molecule detection assay, a nanowell assay, a single molecule enzyme
linked
immunosorbent assay, a direct capture counting assay, etc.
[0068] "Drugs of abuse" is used herein to refer to one or more additive
substances (such as
a drug) taken for non-medical reasons (such as for, example, recreational
and/or mind-
altering effects). Excessive overindulgence, use or dependence of such drugs
of abuse is
often referred to as "substance abuse". Examples of drugs of abuse include
alcohol,
barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (such as
ketamine, mescaline
(peyote), PCP, psilocybin, DMT and/or LSD), methaqualone, opioids,
amphetamines
(including methamphetamines), anabolic steroids, inhalants (namely, substances
which
contain volatile substances that contain psychoactive properties such as, for
example, nitrites,
spray paints, cleaning fluids, markers, glues, etc.) and combinations thereof.
100691 "Dual-specific antibody" is used herein to refer to a full-length
antibody that can
bind two different antigens (or epitopes) in each of its two binding arms (a
pair of HC/LC)
(see PCT publication WO 02/02773). Accordingly, a dual-specific binding
protein has two
identical antigen binding arms, with identical specificity and identical CDR
sequences, and is
bivalent for each antigen to which it binds.
10070] "Dual variable domain" is used herein to refer to two or more antigen
binding sites
on a binding protein, which may be divalent (two antigen binding sites),
tetravalent (four
antigen binding sites), or multivalent binding proteins. DVDs may be
monospecific, i.e.,
capable of binding one antigen (or one specific epitope), or multispecific,
i.e., capable of
binding two or more antigens (i.e., two or more epitopes of the same target
antigen molecule
or two or more epitopes of different target antigens). A preferred DVD binding
protein
comprises two heavy chain DVD polypeptides and two light chain DVD
polypeptides and is
referred to as a "DVD immunoglobulin- or "DVD-Ig." Such a DVD-Ig binding
protein is
thus tetrameric and reminiscent of an IgG molecule but provides more antigen
binding sites
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than an IgG molecule. Thus, each half of a tetrameric DVD-Ig molecule is
reminiscent of
one half of an IgG molecule and comprises a heavy chain DVD polypeptide and a
light chain
DVD polypeptide, but unlike a pair of heavy and light chains of an IgG
molecule that
provides a single antigen binding domain, a pair of heavy and light chains of
a DVD-Ig
provide two or more antigen binding sites.
100711 Each antigen binding site of a DVD-Ig binding protein may be derived
from a donor
("parental") monoclonal antibody and thus comprises a heavy chain variable
domain (VH)
and a light chain variable domain (VL) with a total of six CDRs involved in
antigen binding
per antigen binding site. Accordingly, a DVD-Ig binding protein that binds two
different
epitopes (i.e., two different epitopes of two different antigen molecules or
two different
epitopes of the same antigen molecule) comprises an antigen binding site
derived from a first
parental monoclonal antibody and an antigen binding site of a second parental
monoclonal
antibody.
100721 A description of the design, expression, and characterization of DVD-Ig
binding
molecules is provided in PCT Publication No. WO 2007/024715, U.S. Patent No.
7,612,181,
and Wu et al., Nature Biotech., 25: 1290-1297 (2007). A preferred example of
such DVD-Ig
molecules comprises a heavy chain that comprises the structural formula VD1-
(X1)n-VD2-C-
(X2)n, wherein VD1 is a first heavy chain variable domain, VD2 is a second
heavy chain
variable domain, C is a heavy chain constant domain, X1 is a linker with the
proviso that it is
not CHL X2 is an Pc region, and n is 0 or 1, but preferably 1; and a light
chain that comprises
the structural formula VD1-(Xl)n-VD2-C-(X2)n, wherein VD1 is a first light
chain variable
domain, VD2 is a second light chain variable domain, C is a light chain
constant domain, X1
is a linker with the proviso that it is not CHL and X2 does not comprise an Fc
region; and n
is 0 or 1, but preferably 1. Such a DVD-Ig may comprise two such heavy chains
and two
such light chains, wherein each chain comprises variable domains linked in
tandem without
an intervening constant region between variable regions, wherein a heavy chain
and a light
chain associate to form tandem functional antigen binding sites, and a pair of
heavy and light
chains may associate with another pair of heavy and light chains to form a
tetrameric binding
protein with four functional antigen binding sites. In another example, a DVD-
Ig molecule
may comprise heavy and light chains that each comprise three variable domains
(VD1, VD2,
VD3) linked in tandem without an intervening constant region between variable
domains,
wherein a pair of heavy and light chains may associate to form three antigen
binding sites,
and wherein a pair of heavy and light chains may associate with another pair
of heavy and
light chains to form a tetrameric binding protein with six antigen binding
sites.
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100731 In a preferred embodiment, a DVD-Ig binding protein not only binds the
same target
molecules bound by its parental monoclonal antibodies, but also possesses one
or more
desirable properties of one or more of its parental monoclonal antibodies.
Preferably, such an
additional property is an antibody parameter of one or more of the parental
monoclonal
antibodies. Antibody parameters that may be contributed to a DVD-Ig binding
protein from
one or more of its parental monoclonal antibodies include, but are not limited
to, antigen
specificity, antigen affinity, potency, biological function, epitope
recognition, protein
stability, protein solubility, production efficiency, immunogenicity,
pharmacokinetics,
bioavailability, tissue cross reactivity, and orthologous antigen binding.
100741 A DVD-Ig binding protein binds at least one epitope of UCH-Li. Non-
limiting
examples of a DVD-Ig binding protein include a DVD-Ig binding protein that
binds one or
more epitopes of UCH-L, a DVD-Ig binding protein that binds an epitope of a
human UCH-
Li and an epitope of UCH-L1 of another species (for example, mouse), and a DVD-
Ig
binding protein that binds an epitope of a human UCH-L1 and an epitope of
another target
molecule.
100751 "Dynamic range" as used herein refers to range over which an assay
readout is
proportional to the amount of target molecule or analyte in the sample being
analyzed.
100761 "Epitope," or "epitopes," or "epitopes of interest" refer to a site(s)
on any molecule
that is recognized and can bind to a complementary site(s) on its specific
binding partner.
The molecule and specific binding partner are part of a specific binding pair.
For example,
an epitope can be on a pol ypepti de, a protein, a hapten, a carbohydrate
antigen (such as, but
not limited to, glycolipids, glycoproteins or lipopolysaccharides), or a
polysaccharide. Its
specific binding partner can be, but is not limited to, an antibody.
100771 "Fragment antigen-binding fragment" or "Fab fragment" as used herein
refers to a
fragment of an antibody that binds to antigens and that contains one antigen-
binding site, one
complete light chain, and part of one heavy chain. Fab is a monovalent
fragment consisting
of the VL, VH, CL and CH1 domains. Fab is composed of one constant and one
variable
domain of each of the heavy and the light chain. The variable domain contains
the paratope
(the antigen-binding site), comprising a set of complementarity determining
regions, at the
amino terminal end of the monomer. Each arm of the Y thus binds an epitope on
the antigen.
Fab fragments can be generated such as has been described in the art, e.g.,
using the enzyme
papain, which can be used to cleave an immunoglobulin monomer into two Fab
fragments
and an Fc fragment, or can be produced by recombinant means.
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100781 "F(ab')2 fragment" as used herein refers to antibodies generated by
pepsin digestion
of whole IgG antibodies to remove most of the Fc region while leaving intact
some of the
hinge region. F(ab')2 fragments have two antigen-binding F(ab) portions linked
together by
disulfide bonds, and therefore are divalent with a molecular weight of about
110 kDa.
Divalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG
molecules and
enable a better penetration into tissue thus facilitating better antigen
recognition in
immunohistochemistry. The use of F(ab.)2 fragments also avoids unspecific
binding to Fc
receptor on live cells or to Protein A/G. F(ab')2 fragments can both bind and
precipitate
antigens.
100791 "Framework" (FR) or "Framework sequence" as used herein may mean the
remaining sequences of a variable region minus the CDRs. Because the exact
definition of a
CDR sequence can be determined by different systems (for example, see above),
the meaning
of a framework sequence is subject to correspondingly different
interpretations. The six
CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy
chain) also
divide the framework regions on the light chain and the heavy chain into four
sub-regions
(FR1, FR2, F1(3, and FR4) on each chain, in which CDR1 is positioned between
FR1 and
FR2, CDR2 between F1(2 and F1(3, and CDR3 between FR3 and FR4. Without
specifying
the particular sub-regions as FR1, FR2, FR3, or FR4, a framework region, as
referred by
others, represents the combined FRs within the variable region of a single,
naturally
occurring immunoglobulin chain. As used herein, a FR represents one of the
four sub-
regions, and FRs represents two or more of the four sub-regions constituting a
framework
region.
100801 Human heavy chain and light chain FR sequences are known in the art
that can be
used as heavy chain and light chain "acceptor" framework sequences (or simply,
"acceptor"
sequences) to humanize a non-human antibody using techniques known in the art.
In one
embodiment, human heavy chain and light chain acceptor sequences are selected
from the
framework sequences listed in publicly available databases such as V-base
(hypertext transfer
protocol://vbase.mrc-cpe.cam.ac.uk/) or in the international ImMunoGeneTics0
(IMGT0)
information system (hypertext transfer
protocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).
100811 "Functional antigen binding site" as used herein may mean a site on a
binding
protein (e.g., an antibody) that is capable of binding a target antigen. The
antigen binding
affinity of the antigen binding site may not be as strong as the parent
binding protein, e.g.,
parent antibody, from which the antigen binding site is derived, but the
ability to bind antigen
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must be measurable using any one of a variety of methods known for evaluating
protein, e.g.,
antibody, binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen
binding sites of a multivalent protein, e.g., multivalent antibody, herein
need not be
quantitatively the same.
[0082] "GFAP" is used herein to describe glial fibrillary acidic protein. GFAP
is a protein
that is encoded by the GFAP gene in humans, and which can be produced (e.g.,
by
recombinant means, in other species).
[0083] "GFAP status" can mean either the level or amount of GFAP at a point in
time (such
as with a single measure of GFAP), the level or amount of GFAP associated with
monitoring
(such as with a repeat test on a subject to identify an increase or decrease
in GFAP amount),
the level or amount of GFAP associated with treatment for traumatic brain
injury (whether a
primary brain injury and/or a secondary brain injury) or combinations thereof.
"Glasgow Coma Scale- or "GCS- as used herein refers to a 15-point scale (e.g.,
described in
1974 by Graham Teasdale and Bryan Jennett, Lancet 1974; 2:81-4) that provides
a practical
method for assessing impairment of conscious level in patients who have
suffered a brain
injury. The test measures the best motor response, verbal response and eye
opening response
with these values: I. Best Motor Response (6 - obey 2-part request; 5 - brings
hand above
clavicle to stimulus on head neck; 4 - bends arm at elbow rapidly but features
not
predominantly abnormal; 3 - bends arm at elbow, features clearly predominantly
abnormal; 2
- extends arm at elbow; 1- no movement in arms/legs, no interfering factor; NT
- paralyzed
or other limiting factor); IL Verbal Response (5 - correctly gives name, place
and date; 4 -
not orientated but communication coherently; 3 - intelligible single words; 2 -
only
moans/groans; 1- no audible response, no interfering factor; NT - factor
interfering with
communication); and III. Eye Opening (4 - open before stimulus; 3 - after
spoken or shouted
request; 2 - after fingertip stimulus; 1 - no opening at any time, no
interfering factor; NT -
closed by local factor). The final score is determined by adding the values of
A
subject is considered to have a mild TBI if the GCS score is 13-15. A subject
is considered to
have a moderate TBI if the GCS score is 9-12. A subject is considered to have
a severe TBI
if the GCS score is 8 or less, typically 3-8.
[0084] "Glasgow Outcome Scale" as used herein refers to a global scale for
functional
outcome that rates patient status into one of five categories: Dead,
Vegetative State, Severe
Disability, Moderate Disability or Good Recovery. "Extended Glasgow Outcome
Scale" or
"GOSE" as used interchangeably herein provides more detailed categorization
into eight
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categories by subdividing the categories of severe disability, moderate
disability and good
recovery into a lower and upper category as shown in Table 1.
Table 1
1 Death
2 Vegetative state VX
3 Lower severe disability SD - Condition of unawareness
with only reflex
responses but with periods of spontaneous
4 Upper severe disability SD +
eye opening
Lower moderate Patient who is dependent for
daily support
MD
disability - for mental or physical
disability, usually a
combination of both. If the patient can be
left alone for more than 8 hours at home it is
6 Upper moderate disability MD + upper level of SD, if not
then it is low level
of SD.
Patients have some disability such as
aphasia, hemiparesis or epilepsy and/or
7 Lower good recovery GR -
deficits of memory or personality but are
able to look after themselves. They are
independent at home but dependent outside.
If they are able to return to work even with
8 Upper good recovery GR +
special arrangement it is upper level of MD,
if not then it is low level of MD.
10085] "Humanized antibody" is used herein to describe an antibody that
comprises heavy
and light chain variable region sequences from a non-human species (e.g., a
mouse) but in
which at least a portion of the VH and/or VL sequence has been altered to be
more "human-
like,- i.e., more similar to human germline variable sequences. A "humanized
antibody" is
an antibody or a variant, derivative, analog, or fragment thereof, which
immunospecifically
binds to an antigen of interest and which comprises a framework (FR) region
having
substantially the amino acid sequence of a human antibody and a complementary
determining
region (CDR) having substantially the amino acid sequence of a non-human
antibody. As
used herein, the term "substantially" in the context of a CDR refers to a CDR
having an
amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, or at
least 99% identical to the amino acid sequence of a non-human antibody CDR. A
humanized
antibody comprises substantially all of at least one, and typically two,
variable domains (Fab,
Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions
correspond to
those of a non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the
framework regions are those of a human immunoglobulin consensus sequence. In
an
embodiment, a humanized antibody also comprises at least a portion of an
immunoglobulin
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constant region (Fc), typically that of a human immunoglobulin. In some
embodiments, a
humanized antibody contains the light chain as well as at least the variable
domain of a heavy
chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions
of the
heavy chain. In some embodiments, a humanized antibody only contains a
humanized light
chain. In some embodiments, a humanized antibody only contains a humanized
heavy chain.
In specific embodiments, a humanized antibody only contains a humanized
variable domain
of a light chain and/or humanized heavy chain.
[0086] A humanized antibody can be selected from any class of immunoglobulins,
including IgM, IgG, IgD, IgA, and IgE, and any isotype, including without
limitation IgG1 ,
IgG2, IgG3, and IgG4. A humanized antibody may comprise sequences from more
than one
class or isotype, and particular constant domains may be selected to optimize
desired effector
functions using techniques well-known in the art.
[0087] The framework regions and CDRs of a humanized antibody need not
correspond
precisely to the parental sequences, e.g., the donor antibody CDR or the
consensus
framework may be mutagenized by substitution, insertion, and/or deletion of at
least one
amino acid residue so that the CDR or framework residue at that site does not
correspond to
either the donor antibody or the consensus framework. In a preferred
embodiment, such
mutations, however, will not be extensive. Usually, at least 80%, preferably
at least 85%,
more preferably at least 90%, and most preferably at least 95% of the
humanized antibody
residues will correspond to those of the parental FR and CDR sequences. As
used herein, the
term "consensus framework" refers to the framework region in the consensus
immunoglobulin sequence. As used herein, the term "consensus immunoglobulin
sequence"
refers to the sequence formed from the most frequently occurring amino acids
(or
nucleotides) in a family of related immunoglobulin sequences (see, e.g.,
Winnaker, From
Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A "consensus
immunoglobulin
sequence" may thus comprise a "consensus framework region(s)" and/or a
"consensus
CDR(s)". In a family of inununoglobulins, each position in the consensus
sequence is
occupied by the amino acid occurring most frequently at that position in the
family. If two
amino acids occur equally frequently, either can be included in the consensus
sequence.
[0088] "Identical" or "identity," as used herein in the context of two or more
polypeptide or
polynucleotide sequences, can mean that the sequences have a specified
percentage of
residues that are the same over a specified region. The percentage can be
calculated by
optimally aligning the two sequences, comparing the two sequences over the
specified region,
determining the number of positions at which the identical residue occurs in
both sequences
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to yield the number of matched positions, dividing the number of matched
positions by the
total number of positions in the specified region, and multiplying the result
by 100 to yield
the percentage of sequence identity. In cases where the two sequences are of
different
lengths or the alignment produces one or more staggered ends and the specified
region of
comparison includes only a single sequence, the residues of the single
sequence are included
in the denominator but not the numerator of the calculation.
[0089] "Injury to the head- or "head injury- as used interchangeably herein,
refers to any
trauma to the scalp, skull, or brain. Such injuries may include only a minor
bump on the skull
or may be a serious brain injury. Such injuries include primary injuries to
the brain and/or
secondary injuries to the brain. Primary brain injuries occur during the
initial insult and
result from displacement of the physical structures of the brain. More
specifically, a primary
brain injury is the physical damage to parenchyma (tissue, vessels) that
occurs during the
traumatic event, resulting in shearing and compression of the surrounding
brain tissue.
Secondary brain injuries occur subsequent to the primary injury and may
involve an array of
cellular processes. More specifically, a secondary brain injury refers to the
changes that
evolve over a period of time (from hours to days) after the primary brain
injury. It includes an
entire cascade of cellular, chemical, tissue, or blood vessel changes in the
brain that
contribute to further destruction of brain tissue.
[0090] An injury to the head can be either closed or open (penetrating). A
closed head
injury refers to a trauma to the scalp, skull or brain where there is no
penetration of the skull
by a striking object. An open head injury refers a trauma to the scalp, skull
or brain where
there is penetration of the skull by a striking object. An injury to the head
may be caused by
physical shaking of a person, by blunt impact by an external mechanical or
other force that
results in a closed or open head trauma (e.g., vehicle accident such as with
an automobile,
plane, train, etc.; blow to the head such as with a baseball bat, or from a
firearm), a cerebral
vascular accident (e.g., stroke), one or more falls (e.g., as in sports or
other activities),
explosions or blasts (collectively, "blast injuries") and by other types of
blunt force trauma
Alternatively, an injury to the head may be caused by the ingestion and/or
exposure to a fire,
chemical, toxin or a combination of a chemical and toxin. Examples of such
chemicals
and/or toxins include molds, asbestos, pesticides and insecticides, organic
solvents, paints,
glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organic
metals (such
as methyl mercury, tetraethyl lead and organic tin) and/or one or more drugs
of abuse.
Alternatively, an injury to the head may be caused as a result of a subject
suffering from an
autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral
infection (e.g.,
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SARS-CoV-2), a fungal infection, a bacterial infection, meningitis,
hydrocephalus, or any
combinations thereof. In some cases, it is not possible to be certain whether
any such event
or injury has occurred or taken place. For example, there may be no history on
a patient or
subject, the subject may be unable to speak, the subject may be aware of what
events they
were exposed to, etc. Such circumstances are described herein as the subject
"may have
sustained an injury to the head." In certain embodiments herein, the closed
head injury does
not include and specifically excludes a cerebral vascular accident, such as
stroke.
[0091] "Intracranial lesion" as used herein refers to an area of injury within
the brain. An
intracranial lesion can be an abnormality seen on a CT scan or brain-imaging
test, such as
magnetic resonance imaging (MRI). On CT or MRI scans, brain lesions can appear
as dark
or light spots that do not look like normal brain tissue.
[0092] "Isolated polynucleotide" as used herein may mean a polynucleotide
(e.g., of
genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue
of its origin, the
isolated polynucleolide is not associated with all or a portion of a
polynucleotide with which
the "isolated polynucleotide" is found in nature; is operably linked to a
polynucleotide that it
is not linked to in nature; or does not occur in nature as part of a larger
sequence.
100931 "Label" and "detectable label" as used herein refer to a moiety
attached to an
antibody or an analyte to render the reaction between the antibody and the
analyte detectable,
and the antibody or analyte so labeled is referred to as "detectably labeled."
A label can
produce a signal that is detectable by visual or instrumental means. Various
labels include
signal-producing substances, such as chromagens, fluorescent compounds,
chemiluminescent
compounds, radioactive compounds, and the like. Representative examples of
labels include
moieties that produce light, e.g., acridinium compounds, and moieties that
produce
fluorescence, e.g., fluorescein. Other labels are described herein. In this
regard, the moiety,
itself, may not be detectable but may become detectable upon reaction with yet
another
moiety. Use of the term "detectably labeled" is intended to encompass such
labeling.
[0094] Any suitable detectable label as is known in the art can be used. For
example, the
detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S,
90Y, 99Tc,
111In, 1251, 1311, 177Lu, 166Ho, and 153Sm), an enzymatic label (such as
horseradish
peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the
like), a
chemiluminescent label (such as acridinium esters, thioesters, or
sulfonamides; luminol,
isoluminol, phenanthridinium esters, and the like), a fluorescent label (such
as fluorescein
(e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-
carboxyfluorescein,
6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein
isothiocyanate, and the like)),
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rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc
sulfide-capped
cadmium selenide), a thermometric label, or an immuno-polymerase chain
reaction label. An
introduction to labels, labeling procedures and detection of labels is found
in Polak and Van
Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y.
(1997), and
in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996),
which is a
combined handbook and catalogue published by Molecular Probes, Inc., Eugene,
Oregon. A
fluorescent label can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896,
5,573,904,
5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by
reference in their
entireties). An acridinium compound can be used as a detectable label in a
homogeneous
chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Len.
16: 1324-1328
(2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004);
Adamczyk et al.,
Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett.
5: 3779-3782
(2003)).
100951 In one aspect, the acridinium compound is an acridinium-9-carboxamide.
Methods
for preparing acridinium 9-carboxamides are described in Mattingly, J.
Biolumin.
Chemilumin. 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639
(1998);
Adamczyk et al., Tetrahedron 55: 10899-10914 (1999); Adamczyk et al., Org.
Lett. 1: 779-
781 (1999); Adamczyk et al., Bioconjugate Chem. 11: 714-724 (2000); Mattingly
et al., In
Luminescence Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press:
Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5: 3779-3782
(2003); and U.S.
Patent Nos. 5,468,646, 5,543,524 and 5,783,699 (each of which is incorporated
herein by
reference in its entirety for its teachings regarding same).
100961 Another example of an acridinium compound is an acridinium-9-
carboxylate aryl
ester. An example of an acridinium-9-carboxylate aryl ester of formula II is
10-methy1-9-
(phenoxycarbonyflacridinium fluorosulfonate (available from Cayman Chemical,
Ann Arbor,
MI). Methods for preparing acridinium 9-carboxylate aryl esters are described
in McCapra et
Photochem. Photobiol. 4: 1111-21(1965); Razavi et al., Luminescence 15: 245-
249
(2000); Razavi et al., Luminescence 15: 239-244 (2000); and U.S. Patent No.
5,241,070 (each
of which is incorporated herein by reference in its entirety for its teachings
regarding same).
Such acridinium-9-carboxylate aryl esters are efficient chemiluminescent
indicators for
hydrogen peroxide produced in the oxidation of an analyte by at least one
oxidase in terms of
the intensity of the signal and/or the rapidity of the signal. The course of
the
chemiluminescent emission for the acridinium-9-carboxylate aryl ester is
completed rapidly,
i.e., in under 1 second, while the acridinium-9-carboxamide chemiluminescent
emission
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extends over 2 seconds. Acridinium-9-carboxylate aryl ester, however, loses
its
chemiluminescent properties in the presence of protein. Therefore, its use
requires the
absence of protein during signal generation and detection. Methods for
separating or
removing proteins in the sample are well-known to those skilled in the art and
include, but
are not limited to, ultrafiltration, extraction, precipitation, dialysis,
chromatography, and/or
digestion (see, e.g., Wells, High Throughput Bioanalytical Sample Preparation.
Methods and
Automation Strategies, Elsevier (2003)). The amount of protein removed or
separated from
the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Further
details
regarding acridinium-9-carboxylate aryl ester and its use are set forth in
U.S. Patent App. No.
11/697,835, filed April 9, 2007. Acridinium-9-carboxylate aryl esters can be
dissolved in any
suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or
aqueous
sodium cholate.
100971 "Linking sequence- or -linking peptide sequence" refers to a natural or
artificial
polypeptide sequence that is connected to one or more polypeptide sequences of
interest (e.g.,
full-length, fragments, etc.). The term -connected" refers to the joining of
the linking
sequence to the polypeptide sequence of interest. Such polypeptide sequences
are preferably
joined by one or more peptide bonds. Linking sequences can have a length of
from about 4
to about 50 amino acids. Preferably, the length of the linking sequence is
from about 6 to
about 30 amino acids. Natural linking sequences can be modified by amino acid
substitutions, additions, or deletions to create artificial linking sequences.
Linking sequences
can be used for many purposes, including in recombinant Fabs. Exemplary
linking sequences
include, but are not limited to: (i) Histidine (His) tags, such as a 6X His
tag, which has an
amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences
to
facilitate the isolation and purification of polypeptides and antibodies of
interest; (ii)
Enterokinase cleavage sites, like His tags, are used in the isolation and
purification of
proteins and antibodies of interest. Often, enterokinase cleavage sites are
used together with
His tags in the isolation and purification of proteins and antibodies of
interest. Various
enterokinase cleavage sites are known in the art. Examples of enterokinase
cleavage sites
include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO:
4) and
derivatives thereof (e.g., ADDDDK (SEQ ID NO: 5), etc.); (iii) Miscellaneous
sequences can
be used to link or connect the light and/or heavy chain variable regions of
single chain
variable region fragments. Examples of other linking sequences can be found in
Bird et al.,
Science 242: 423-426 (1988); Huston et al., PNAS USA 85: 5879-5883 (1988); and
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McCafferty et al., Nature 348: 552-554 (1990). Linking sequences also can be
modified for
additional functions, such as attachment of drugs or attachment to solid
supports. In the
context of the present disclosure, the monoclonal antibody, for example, can
contain a linking
sequence, such as a His tag, an enterokinase cleavage site, or both.
10098] "Magnetic resonance imaging" or "MRI" as used interchangeably herein
refers to a
medical imaging technique used in radiology to fonia pictures of the anatomy
and the
physiological processes of the body in both health and disease (e.g., referred
to herein
interchangeably as "an MRI", "an MRI procedure- or "an MRI scan"). MRI is a
form of
medical imaging that measures the response of the atomic nuclei of body
tissues to high-
frequency radio waves when placed in a strong magnetic field, and that
produces images of
the internal organs. MRI scanners, which is based on the science of nuclear
magnetic
resonance (NMR), use strong magnetic fields, radio waves, and field gradients
to generate
images of the inside of the body.
100991 "Monoclonal antibody- as used herein refers to an antibody obtained
from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies
comprising the population are identical except for possible naturally
occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly specific,
being directed
against a single antigen. Furthermore, in contrast to polyclonal antibody
preparations that
typically include different antibodies directed against different determinants
(epitopes), each
monoclonal antibody is directed against a single determinant on the antigen.
'[he monoclonal
antibodies herein specifically include "chimeric" antibodies in which a
portion of the heavy
and/or light chain is identical with or homologous to corresponding sequences
in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass, while
the remainder of the chain(s) is identical with or homologous to corresponding
sequences in
antibodies derived from another species or belonging to another antibody class
or subclass, as
well as fragments of such antibodies, so long as they exhibit the desired
biological.
[0100] "Multivalent binding protein" is used herein to refer to a binding
protein comprising
two or more antigen binding sites (also referred to herein as "antigen binding
domains"). A
multivalent binding protein is preferably engineered to have three or more
antigen binding
sites and is generally not a naturally occurring antibody. The term
"multispecific binding
protein" refers to a binding protein that can bind two or more related or
unrelated targets,
including a binding protein capable of binding two or more different epitopes
of the same
target molecule.
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[0101] "Negative predictive value or "NPV" as used interchangeably herein
refers to the
probability that a subject has a negative outcome given that they have a
negative test result.
[0102] "Reference level" as used herein refers to an assay cutoff value that
is used to assess
diagnostic, prognostic, or therapeutic efficacy and that has been linked or is
associated herein
with various clinical parameters (e.g., presence of disease, stage of disease,
severity of
disease, progression, non-progression, or improvement of disease, etc.). An
"absolute
amount- as used herein refers to the absolute value of a change or difference
between at least
two assay results taken or sampled at different time points and, which similar
to a reference
level, has been linked or is associated herein with various clinical
parameters (e.g., presence
of disease, stage of disease, severity of disease, progression, non-
progression, or
improvement of disease, etc.). "Absolute value" as used herein refers to the
magnitude of a
real number (such as, for example, the difference between two compared levels
(such as
levels taken at a first time point and levels taken at a second time point))
without regard to its
sign, i.e., regardless of whether it is positive or negative.
[0103] This disclosure provides exemplary reference levels and absolute
amounts (e.g.,
calculated by comparing reference levels at different time points). However,
it is well-known
that reference levels and absolute amounts may vary depending on the nature of
the
immunoassay (e.g., antibodies employed, reaction conditions, sample purity,
etc.) and that
assays can be compared and standardized. It further is well within the
ordinary skill of one in
the art to adapt the disclosure herein for other immunoassays to obtain
immunoassay-specific
reference levels and absolute amounts for those other immunoassays based on
the description
provided by this disclosure. Whereas the precise value of the reference level
and absolute
amount may vary between assays, the findings as described herein should be
generally
applicable and capable of being extrapolated to other assays.
[0104] "Point-of-care device" refers to a device used to provide medical
diagnostic testing
at or near the point-of-care (namely, outside of a laboratory), at the time
and place of patient
care (such as in a hospital, physician's office, urgent or other medical care
facility, a patient's
home, a nursing home and/or a long term care and/or hospice facility).
Examples of point-of-
care devices include those produced by Abbott Laboratories (Abbott Park, IL)
(e.g., i-STAT
and i-STAT Alinity, Universal Biosensors (Row ville, Australia) (see US
2006/0134713),
Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles,
USA).
[0105] "Positive predictive value" or "PPV" as used interchangeably herein
refers to the
probability that a subject has a positive outcome given that they have a
positive test result.
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101061 "Quality control reagents" in the context of immunoassays and kits
described herein,
include, but are not limited to, calibrators, controls, and sensitivity
panels. A "calibrator" or
"standard- typically is used (e.g., one or more, such as a plurality) in order
to establish
calibration (standard) curves for interpolation of the concentration of an
analyte, such as an
antibody or an analyte. Alternatively, a single calibrator, which is near a
reference level or
control level (e.g., "low", "medium", or "high" levels), can be used. Multiple
calibrators
(i.e., more than one calibrator or a varying amount of calibrator(s)) can be
used in
conjunction to comprise a "sensitivity panel."
101071 A "receiver operating characteristic" curve or "ROC" curve refers to a
graphical plot
that illustrates the performance of a binary classifier system as its
discrimination threshold is
varied. For example, a ROC curve can be a plot of the true positive rate
against the false
positive rate for the different possible cutoff points of a diagnostic test.
It is created by
plotting the fraction of true positives out of the positives (TPR = true
positive rate) vs. the
fraction of false positives out of the negatives (FPR = false positive rate),
at various threshold
settings. TPR is also known as sensitivity, and FPR is one minus the
specificity or true
negative rate. The ROC curve demonstrates the tradeoff between sensitivity and
specificity
(any increase in sensitivity will be accompanied by a decrease in
specificity); the closer the
curve follows the left-hand border and then the top border of the ROC space,
the more
accurate the test; the closer the curve comes to the 45-degree diagonal of the
ROC space, the
less accurate the test; the slope of the tangent line at a cutoff point gives
the likelihood ratio
(LR) for that value of the test; and the area under the curve is a measure of
test accuracy.
101081 "Recombinant antibody" and "recombinant antibodies refer to antibodies
prepared
by one or more steps, including cloning nucleic acid sequences encoding all or
a part of one
or more monoclonal antibodies into an appropriate expression vector by
recombinant
techniques and subsequently expressing the antibody in an appropriate host
cell. The terms
include, but are not limited to, recombinantly produced monoclonal antibodies,
chimeric
antibodies, humanized antibodies (fully or partially humanized), multi-
specific or multi-
valent structures formed from antibody fragments, bifunctional antibodies,
heteroconjugate
Abs, DVD-Ig0s, and other antibodies as described in (i) herein. (Dual-variable
domain
immunoglobulins and methods for making them are described in Wu, C., et al.,
Nature
Biotechnology, 25:1290-1297 (2007)). The term "bifunctional antibody," as used
herein,
refers to an antibody that comprises a first arm having a specificity for one
antigenic site and
a second arm having a specificity for a different antigenic site, i.e., the
bifunctional antibodies
have a dual specificity.
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101091 "Risk assessment," "risk classification," "risk identification," or
"risk stratification"
of subjects (e.g., patients) as used herein refers to the evaluation of
factors including
biomarkers, to predict the risk of occurrence of future events including
disease onset or
disease progression, so that treatment decisions regarding the subject may be
made on a more
informed basis.
101101 "Sample," "test sample," "specimen," "sample from a subject,"
"biological sample,"
and "patient sample- as used interchangeably herein may be a sample of blood,
such as whole
blood (including for example, capillary blood, venous blood, dried blood spot,
etc.), serum or
plasma, or tissue, saliva, urineõ amniotic fluid, an oropharyngeal specimen, a
nasopharyngeal specimens, lower respiratory specimens such as, but not limited
to, sputum,
endotracheal aspirate or bronchoalveolar lavage, cerebrospinal fluid,
placental cells or tissue,
endothelial cells, leukocytes, or monocytes. The sample can be used directly
as obtained
from a patient or can be pre-treated, such as by filtration, distillation,
extraction,
concentration, centrifugation, inactivation of interfering components,
addition of reagents,
and the like, to modify the character of the sample in some manner as
discussed herein or
otherwise as is known in the art. Additionally, the sample can be a
nasopharyngeal or
oropharyngeal sample obtained using one or more swabs that, once obtained, is
placed in a
sterile tube containing a virus transport media (VTM) or universal transport
media (UTM),
and retained therein or transferred to another media for testing.
101111 A variety of cell types, tissue, or bodily fluid may be utilized to
obtain a sample.
Such cell types, tissues, and fluid may include sections of tissues such as
biopsy and autopsy
samples, oropharyngeal specimens, nasopharyngeal specimens, frozen sections
taken for
histologic purposes, blood (such as whole blood, dried blood spots, etc.),
plasma, serum,
saliva, red blood cells, platelets, interstitial fluid, cerebral spinal fluid,
etc. Cell types and
tissues may also include lymph fluid, cerebrospinal fluid, or any fluid
collected by aspiration.
A tissue or cell type may be provided by removing a sample of cells from a
human and a non-
human animal but can also be accomplished by using previously isolated cells
(e.g., isolated
by another person, at another time, and/or for another purpose). Archival
tissues, such as
those having treatment or outcome history, may also be used. Protein or
nucleotide isolation
and/or purification may not be necessary. In some embodiments, the sample is a
blood
sample (e.g., a whole blood sample, a serum sample, or a plasma sample). In
some
embodiments, the sample is a whole blood sample. In some embodiments. the
sample is a
capillary blood sample. In some embodiments, the sample is a dried blood spot.
In some
embodiments, the sample is a serum sample. In yet other embodiments, the
sample is a
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plasma sample. In some embodiments, the sample is an oropharyngeal specimen.
In other
embodiments, the sample is a nasopharyngeal specimen. In other embodiments,
the sample is
sputum. In other embodiments, the sample is endotracheal aspirate. In still
yet other
embodiments, the sample is bronchoalveolar lavage. In still yet other
embodiments, the
sample is a saliva sample.
[0112] "Sensitivity" of an assay as used herein refers to the
proportion of subjects for
whom the outcome is positive that are correctly identified as positive (e.g.,
correctly
identifying those subjects with a disease or medical condition for which they
are being
tested). For example, this might include correctly identifying subjects as
having a TBI as
distinct from those who do not have a TBI, correctly identifying subjects
having a moderate,
severe, or moderate to severe TBI as distinct from those having a mild TBI,
correctly
identifying subjects as having a mild TBI as distinct from those having a
moderate, severe, or
moderate to severe TBI, correctly identifying subjects as having a moderate,
severe, or
moderate to severe TBI as distinct from those having no TBI or correctly
identifying subjects
as having a mild TBI as distinct from those having no TBI etc..
[0113] "Specificity" of an assay as used herein refers to the proportion of
subjects for
whom the outcome is negative that are correctly identified as negative (e.g.,
correctly
identifying those subjects who do not have a disease or medical condition for
which they are
being tested). For example, this might include correctly identifying subjects
not having an
1131 as distinct from those who do have a TBI, correctly identifying subjects
not having a
moderate, severe, or moderate to severe TBI as distinct from those having a
mild TBI,
correctly identifying subjects as not having a mild TBI as distinct from those
having a
moderate, severe, or moderate to severe TBI, etc.).
[0114] "Series of calibrating compositions" refers to a plurality of
compositions
comprising a known concentration of UCH-L1, wherein each of the compositions
differs
from the other compositions in the series by the concentration of UCH-Li.
[0115] "Solid phase" or "solid support" as used interchangeably herein, refers
to any
material that can be used to attach and/or attract and immobilize (1) one or
more capture
agents or capture specific binding partners, or (2) one or more detection
agents or detection
specific binding partners. The solid phase can be chosen for its intrinsic
ability to attract and
immobilize a capture agent. Alternatively, the solid phase can have affixed
thereto a linking
agent that has the ability to attract and immobilize the (1) capture agent or
capture specific
binding partner, or (2) detection agent or detection specific binding partner.
For example, the
linking agent can include a charged substance that is oppositely charged with
respect to the
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capture agent (e.g., capture specific binding partner) or detection agent
(e.g., detection
specific binding partner) itself or to a charged substance conjugated to the
(1) capture agent
or capture specific binding partner or (2) detection agent or detection
specific binding partner.
In general, the linking agent can be any binding partner (preferably specific)
that is
immobilized on (attached to) the solid phase and that has the ability to
immobilize the (1)
capture agent or capture specific binding partner, or (2) detection agent or
detection specific
binding partner through a binding reaction. The linking agent enables the
indirect binding of
the capture agent to a solid phase material before the performance of the
assay or during the
performance of the assay. For examples, the solid phase can be plastic,
derivatized plastic,
magnetic, or non-magnetic metal, glass or silicon, including, for example, a
test tube,
microtiter well, sheet, bead, microparticle, chip, and other configurations
known to those of
ordinary skill in the art.
[0116] "Specific binding- or "specifically binding- as used herein may refer
to the
interaction of an antibody, a protein, or a peptide with a second chemical
species, wherein the
interaction is dependent upon the presence of a particular structure (e.g., an
antigenic
determinant or epitope) on the chemical species; for example, an antibody
recognizes and
binds to a specific protein structure rather than to proteins generally. If an
antibody is
specific for epitope "A", the presence of a molecule containing epitope A (or
free, unlabeled
A), in a reaction containing labeled "A" and the antibody, will reduce the
amount of labeled
A bound to the antibody.
[0117] "Specific binding partner" is a member of a specific binding pair. A
specific
binding pair comprises two different molecules, which specifically bind to
each other through
chemical or physical means. Therefore, in addition to antigen and antibody
specific binding
pairs of common immunoassays, other specific binding pairs can include biotin
and avidin (or
streptavidin), carbohydrates and lectins, complementary nucleotide sequences,
effector and
receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and
the like.
Furthermore, specific binding pairs can include members that are analogs of
the original
specific binding members, for example, an analyte-analog. Immunoreactive
specific binding
members include antigens, antigen fragments, and antibodies, including
monoclonal and
polyclonal antibodies as well as complexes and fragments thereof, whether
isolated or
recombinantly produced.
[0118] "Statistically significant" as used herein refers to the likelihood
that a relationship
between two or more variables is caused by something other than random chance.
Statistical
hypothesis testing is used to determine whether the result of a data set is
statistically
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significant. In statistical hypothesis testing, a statistically significant
result is attained
whenever the observed p-value of a test statistic is less than the
significance level defined of
the study. The p-value is the probability of obtaining results at least as
extreme as those
observed, given that the null hypothesis is true. Examples of statistical
hypothesis analysis
include Wilcoxon signed-rank test, t-test, Chi-Square or Fisher's exact test.
"Significant" as
used herein refers to a change that has not been determined to be
statistically significant (e.g.,
it may not have been subject to statistical hypothesis testing).
101191 "Subject" and "patient" as used herein interchangeably refers to any
vertebrate,
including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse,
goat, rabbit,
sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate
(for example, a
monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human).
In some
embodiments, the subject may be a human or a non-human. In some embodiments,
the
subject is a human. The subject or patient may be undergoing other forms of
treatment.
101201 "Treat," "treating" or "treatment" are each used interchangeably herein
to describe
reversing, alleviating, or inhibiting the progress of a disease and/or injury,
or one or more
symptoms of such disease, to which such term applies. Depending on the
condition of the
subject, the term also refers to preventing a disease, and includes preventing
the onset of a
disease, or preventing the symptoms associated with a disease. A treatment may
be either
performed in an acute or chronic way_ The term also refers to reducing the
severity of a
disease or symptoms associated with such disease prior to affliction with the
disease. Such
prevention or reduction of the severity of a disease prior to affliction
refers to administration
of a pharmaceutical composition to a subject that is not at the time of
administration afflicted
with the disease. "Preventing" also refers to preventing the recurrence of a
disease or of one
or more symptoms associated with such disease. "Treatment" and
"therapeutically," refer to
the act of treating, as "treating" is defined above.
10121] "Traumatic Brain Injury" or "TBI" as used interchangeably herein refers
to a
complex injury with a broad spectrum of symptoms and disabilities. TBI is most
often an
acute event similar to other injuries. TBI can be classified as "mild,"
"moderate," or
"severe." The causes of TBI are diverse and include, for example, physical
shaking by a
person, a car accident, injuries from firearms, cerebral vascular accidents
(e.g., strokes), falls,
explosions or blasts and other types of blunt force trauma. Other causes of
TBI include the
ingestion and/or exposure to one or more fires, chemicals or toxins (such as
molds, asbestos,
pesticides and insecticides, organic solvents, paints, glues, gases (such as
carbon monoxide,
hydrogen sulfide, and cyanide), organic metals (such as methyl mercury,
tetraethyl lead and
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organic tin), one or more drugs of abuse or combinations thereof).
Alternatively, TBI can
occur in subjects suffering from an autoimmune disease, a metabolic disorder,
a brain tumor,
hypoxia, a viral infection (e.g., SARS-CoV-2, meningitis, etc.), fungal
infection (e.g.,
meningitis), bacterial infection (e.g., meningitis), or any combinations
thereof. Young adults
and the elderly are the age groups at highest risk for TBI. In certain
embodiments herein,
traumatic brain injury or TBI does not include and specifically excludes
cerebral vascular
accidents such as strokes.
[0122] "Mild TBI- as used herein refers to a head injury where a subject may
or may not
experience a loss of consciousness. For subjects that experience a loss of
consciousness, it is
typically brief, usually lasting only a few seconds or minutes. Mild TBI is
also referred to as
a concussion, minor head trauma, minor TBI, minor brain injury, and minor head
injury.
While MRI and CT scans are often normal, the individual with mild TBI may have
cognitive
problems such as headache, difficulty thinking, memory problems, attention
deficits, mood
swings and frustration.
[0123] Mild TBI is the most prevalent TBI and is often missed at time of
initial injury.
Typically, a subject has a Glasgow Coma scale number of between 13-15 (such as
13-15 or
14-15). Fifteen percent (15%) of people with mild TBI have symptoms that last
3 months or
more. Common symptoms of mild TBI include fatigue, headaches, visual
disturbances,
memory loss, poor attention/concentration, sleep disturbances, dizziness/loss
of balance,
irritability-emotional disturbances, feelings of depression, and seizures.
Other symptoms
associated with mild TBI include nausea, loss of smell, sensitivity to light
and sounds, mood
changes, getting lost or confused, and/or slowness in thinking.
[0124] "Moderate TBI" as used herein refers to a brain injury where loss of
consciousness
and/or confusion and disorientation is between 1 and 24 hours and the subject
has a Glasgow
Coma scale number of between 9-13 (such as 9-12 or 9-13). The individual with
moderate
TBI may have abnormal brain imaging results. "Severe TBI" as used herein
refers to a brain
injury where loss of consciousness is more than 24 hours and memory loss after
the injury or
penetrating skull injury longer than 24 hours and the subject has a Glasgow
Coma scale
number between 3-8. The deficits range from impairment of higher level
cognitive functions
to comatose states. Survivors may have limited function of arms or legs,
abnormal speech or
language, loss of thinking ability or emotional problems. Individuals with
severe injuries can
be left in long-term unresponsive states. For many people with severe TBI,
long-term
rehabilitation is often necessary to maximize function and independence.
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[0125] "Moderate to severe" TBI as used herein refers to a spectrum of brain
injury that
includes a change from moderate to severe TBI over time and thus encompasses
(e.g.,
temporally) moderate TB1 alone, severe TB1 alone, and moderate to severe TB!
combined.
For example, in some clinical situations, a subject may initially be diagnosed
as having a
moderate TBI but who, over the course of time (minutes, hours or days),
progresses to having
a severe TBI (such, as for example, in situations when there is a brain
bleed). Alternatively,
in some clinical situations, a subject may initially be diagnosed as having a
severe TBI but
who, over the course of time (minutes, hours or days), progresses to having a
moderate TBI.
Such subjects would be examples of patients that could be classified as
"moderate to severe".
Common symptoms of moderate to severe TBI include cognitive deficits including
difficulties with attention, concentration, distractibility, memory, speed of
processing,
confusion, perseveration, impulsiveness, language processing, and/or
"executive functions",
not understanding the spoken word (receptive aphasia), difficulty speaking and
being
understood (expressive aphasia), slurred speech, speaking very fast or very
slow, problems
reading, problems writing, difficulties with interpretation of touch,
temperature, movement,
limb position and fine discrimination, the integration or patterning of
sensory impressions
into psychologically meaningful data, partial or total loss of vision,
weakness of eye muscles
and double vision (diplopia), blurred vision, problems judging distance,
involuntary eye
movements (nystagmus), intolerance of light (photophobia), hearing issues,
such as decrease
or loss of hearing, ringing in the ears (tinnitus), increased sensitivity to
sounds, loss or
diminished sense of smell (anosmia), loss or diminished sense of taste, the
convulsions
associated with epilepsy that can be several types and can involve disruption
in
consciousness, sensory perception, or motor movements, problems with control
of bowel and
bladder, sleep disorders, loss of stamina, appetite changes, problems with
regulation of body
temperature, menstrual difficulties, dependent behaviors, issues with
emotional ability or
stability, lack of motivation, irritability, aggression, depression,
disinhibition, or denial/lack
of awareness. Subjects having a moderate to severe TBI can have a Glasgow Coma
scale
score from 3-12 (which includes the range of 9-12 for a moderate TBI, and 3-8
for a severe
TBI).
[0126] "Ubiquitin carboxy-terminal hydrolase Li" or "UCH-Li" as used
interchangeably
herein refers to a deubiquitinating enzyme encoded by the UCH-LI gene in
humans. UCH-
Li, also known as ubiquitin carboxyl-terminal esterase Li and ubiquitin
thiolesterase, is a
member of a gene family whose products hydrolyze small C-terminal adducts of
ubiquitin to
generate the ubiquitin monomer.
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101271 "UCH-L1 status" can mean either the level or amount of UCH-L1 at a
point in time
(such as with a single measure of UCH-L1), the level or amount of UCH-L1
associated with
monitoring (such as with a repeat test on a subject to identify an increase or
decrease in
UCH-L1 amount), the level or amount of UCH-L1 associated with treatment for
traumatic
brain injury (whether a primary brain injury and/or a secondary brain injury)
or combinations
thereof.
101281 "Variant- is used herein to describe a peptide or polypeptide that
differs in amino
acid sequence by the insertion, deletion, or conservative substitution of
amino acids, but
retain at least one biological activity. Representative examples of
"biological activity"
include the ability to be bound by a specific antibody or to promote an immune
response.
Variant is also used herein to describe a protein with an amino acid sequence
that is
substantially identical to a referenced protein with an amino acid sequence
that retains at least
one biological activity. A conservative substitution of an amino acid, i.e.,
replacing an amino
acid with a different amino acid of similar properties (e.g., hydrophilicity,
degree, and
distribution of charged regions) is recognized in the art as typically
involving a minor change.
These minor changes can be identified, in part, by considering the hydropathic
index of
amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132
(1982). The
hydropathic index of an amino acid is based on a consideration of its
hydrophobicity and
charge. It is known in the art that amino acids of similar hydropathic indexes
can be
substituted and still retain protein function. In one aspect, amino acids
having hydropathic
indexes of 2 are substituted. The hydrophilicity of amino acids can also be
used to reveal
substitutions that would result in proteins retaining biological function. A
consideration of
the hydrophilicity of amino acids in the context of a peptide permits
calculation of the
greatest local average hydrophilicity of that peptide, a useful measure that
has been reported
to correlate well with antigenicity and immunogenicity. U.S. Patent No.
4,554,101,
incorporated fully herein by reference. Substitution of amino acids having
similar
hydrophilicity values can result in peptides retaining biological activity,
for example
immunogenicity, as is understood in the art. Substitutions may be performed
with amino
acids having hydrophilicity values within 2 of each other. Both the
hydrophobicity index
and the hydrophilicity value of amino acids are influenced by the particular
side chain of that
amino acid. Consistent with that observation, amino acid substitutions that
are compatible
with biological function are understood to depend on the relative similarity
of the amino
acids, and particularly the side chains of those amino acids, as revealed by
the
hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant"
also can be used
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to refer to an antigenically reactive fragment of an anti-UCH-L1 antibody that
differs from
the corresponding fragment of anti-UCH-L1 antibody in amino acid sequence but
is still
antigenically reactive and can compete with the corresponding fragment of anti-
UCH-L1
antibody for binding with UCH-Li. "Variant" also can be used to describe a
polypeptide or a
fragment thereof that has been differentially processed, such as by
proteolysis,
phosphorylation, or other post-translational modification, yet retains its
antigen reactivity.
101291 "Vector- is used herein to describe a nucleic acid molecule that can
transport
another nucleic acid to which it has been linked. One type of vector is a
"plasmid", which
refers to a circular double-stranded DNA loop into which additional DNA
segments may be
ligated. Another type of vector is a viral vector, wherein additional DNA
segments may be
ligated into the viral genome. Certain vectors can replicate autonomously in a
host cell into
which they are introduced (e.g., bacterial vectors having a bacterial origin
of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) can be
integrated into the genome of a host cell upon introduction into the host
cell, and thereby are
replicated along with the host genome. Moreover, certain vectors are capable
of directing the
expression of genes to which they are operatively linked. Such vectors are
referred to herein
as "recombinant expression vectors" (or simply, "expression vectors"). In
general, expression
vectors of utility in recombinant DNA techniques are often in the form of
plasmids.
"Plasmid" and "vector" may be used interchangeably as the plasmid is the most
commonly
used form of vector. However, other forms of expression vectors, such as viral
vectors (e.g.,
replication defective retrovi ruses, adenovi ruses and adeno-associated
viruses), which serve
equivalent functions, can be used. In this regard, RNA versions of vectors
(including RNA
viral vectors) may also find use in the context of the present disclosure.
101301 Unless otherwise defined herein, scientific and technical terms used in
connection
with the present disclosure shall have the meanings that are commonly
understood by those
of ordinary skill in the art. For example, any nomenclatures used in
connection with,
and techniques of, cell and tissue culture, molecular biology, immunology,
microbiology,
genetics and protein and nucleic acid chemistry and hybridization described
herein are
those that are well known and commonly used in the art. The meaning and scope
of the
terms should be clear; in the event, however of any latent ambiguity,
definitions provided
herein take precedent over any dictionary or extrinsic definition. Further,
unless otherwise
required by context, singular terms shall include pluralities and plural terms
shall include the
singular.
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2.
Methods of Aiding in the Determination of a Traumatic Brain Injury (TBI)
in a
Subject having a Head Computerized Tomography (CT) Scan that is Negative for a
TBI
[0131] The present disclosure relates, among other methods, to a method of
aiding in
determining whether a subject, such as a human subject, who has sustained, may
have
sustained, or is suspected of sustaining an injury to the head has more likely
than not,
sustained a traumatic brain injury (TBI) where the subject has received one or
more head CT
scans that are negative for a TBI. Specifically, such a method can comprise
the steps of: (a)
performing, simultaneously or sequentially (in any order): (1) at least one
assay on a sample
obtained from the subject within about 24 hours after an actual or suspected
injury to the head
to measure or detect a level of a biomarker in the sample, said biomarker
comprising
ubiquitin carboxy-terminal hydrolase Li (UCH-L1), glial fibrillary acidic
protein (GFAP), or
a combination thereof; and (2) at least one head CT scan on the subject,
within a clinically-
relevant time frame; and (b) diagnosing the subject as more likely than not as
having TBI if
the level of the biomarker is higher than a reference level and the head CT
scan is negative
for a TBI. The sample can be a biological sample.
101321 In yet another aspect, the present disclosure relates to a method of
aiding in
determining whether a subject, such as a human subject, who has sustained, may
have
sustained, or is suspected of sustaining an injury to the head. Specifically,
said method
comprises performing an assay on a sample obtained from the subject within
about 24 hours
after an actual or suspected injury to the head to measure or detect a level
of a biomarker in
the sample, said biomarker comprising ubiquitin carboxy-terminal hydrolase Li
(UCH-L1),
glial fibrillary acidic protein (GFAP), or a combination thereof and where the
method
comprises diagnosing the subject as more likely than not as having traumatic
brain injury
(TBI) if the level of the biomarker is higher than a reference level, and
either a head
computerized tomography (CT) scan on the subject within a clinically-relevant
time frame is
negative for a TBI, or no head CT scan is performed on the subject. The sample
can be a
biological sample. In some aspects, a head CT scan is performed on the
subject. In other
aspects, no head CT scan is performed on the subject.
[0133] As mentioned herein, the at least one assay and, optionally, if
performed, at least one
head CT scan can be performed simultaneously or sequentially in any order. If
performed
sequentially, the assay and the head CT scan can be performed within a
clinically-relevant
time frame, such as for example, within about 1 minute of each other, within
about 2 minutes
of each other, within about 3 minutes of each other, within about 4 minutes of
each other,
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within about 5 minutes of each other, within about 10 minutes of each other,
within about 15
minutes of each other, within 20 minutes of each other, within about 25
minutes of each
other, within about 30 minutes of each other, within about 45 minutes of each
other, within
about 50 minutes of each other, within about 60 minutes of each other, within
about 1 hour of
each other, within about 1.5 hours of each other, within about 2 hours of each
other, within
about 3 hours of each other, within about 4 hours of each other, within about
5 hours of each
other, within about 6 hours of each other, within about 7 hours of each other,
within about 8
hours of each other, within about 9 hours of each other, within about 10 hours
of each other,
within about 11 hours of each other, or within about 12 hours of each other.
101341 In some aspects, the methods described herein allow for the
identification of
subjects who have sustained or may have sustained an injury to the head as
having a TBI
based on one or more biomarker levels in certain instances where such subjects
have received
a head CT scan that is negative for a TBI. The methods described herein allow
for the
identification of subjects who have suffered a TBI but who may otherwise have
been
incorrectly diagnosed as not having a TBI if such diagnosis was based solely
on the result of
one or more head CT scans.
101351 In some embodiments, the method can include obtaining a sample within
about 24
hours of a suspected injury to the subject and contacting the sample with an
antibody for a
biomarker of TBI, such as ubiquitin carboxy-terminal hydrolase Li (UCH-L1),
glial fibrillary
acidic protein (CiPAY), or a combination thereof, to allow formation of a
complex of the
antibody and the biomarker. The method also includes detecting the resulting
antibody-
biomarker complex.
101361 In some embodiments, the sample is taken from the subject, such as a
human
subject, within about 24 hours of injury (e.g., an actual injury) or suspected
injury to the
head, such as within about 0 to about 6 hours, within about 0 to about 8
hours, within about 0
to about 10 hours, within about 0 to about 12 hours, within about 0 to about
18 hours, within
about 6 hours to about 12 hours, within about 6 hours to about 18 hours, or
within about 12
hours to about 18 hours. For example, the sample can be taken from the
subject, such as a
human subject, within about 0 minutes, about 30 minutes, about 60 minutes,
about 90
minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about
6 hours, 7
hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 13
hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about
18 hours, about
19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or
about 24 hours
of injury or suspected injury to the head. In some embodiments, the onset of
the presence of
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the biomarker, such as UCH-L1, GFAP, or a combination thereof, appears within
about 0
minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120
minutes, about 3
hours, about 4 hours, about 5 hours, about 6 hours, 7 hours, about 8 hours,
about 9 hours,
about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14
hours, about 15
hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about
20 hours, about
21 hours, about 22 hours, about 23 hours, or about 24 hours after injury or
suspected injury to
the head.
[0137] Generally, a reference level of the biomarker, such as UCH-LI, GFAP, or
a
combination thereof, can be employed as a benchmark against which to assess
results
obtained upon assaying a test sample for UCH-Ll. Generally, in making such a
comparison,
the reference level of the biomarker, such as UCH-L1, GFAP, or a combination
thereof, is
obtained by running a particular assay a sufficient number of times and under
appropriate
conditions such that a linkage or association of analyte presence, amount or
concentration
with a particular stage or endpoint of TBI or with particular indicia can be
made. Typically,
the reference level of the biomarker, such as UCH-L1, GFAP, or a combination
thereof, is
obtained with assays of reference subjects (or populations of subjects). The
biomarker, such
as UCH-L1, GFAP, or a combination thereof, measured can include fragments
thereof,
degradation products thereof, and/or enzymatic cleavage products thereof.
[0138] In some embodiments, the reference level UCH-L1 is between about 150
pg/mL to
about 700 pg/mL, about 160 pg/mL to about 700 pg/mL, about 170 pg/mL to about
700
pg/mL, about 180 pg/mL to about 700 pg/mL, about 190 pg/mL to about 700 pg/mL,
about
200 pg/mL to about 700 pg/mL, about 150 pg/mL to about 700 pg/mL, about 160
pg/mL to
about 600 pg/mL, about 170 pg/mL to about 600 pg/mL, about 180 pg/mL to about
600
pg/mL, about 190 pg/mL to about 600 pg/mL, about 200 pg/mL to about 600 pg/mL,
about
150 pg/mL to about 500 pg/mL, about 160 pg/mL to about 500 pg/mL, about 170
pg/mL to
about 500 pg/mL, about 180 pg/mL to about 500 pg/mL, about 190 pg/mL to about
500
pg/mL, about 200 pg/mL to about 500 pg/mL, about 150 pg/mL to about 400 pg/mL,
about
160 pg/mL to about 400 pg/mL, about 170 pg/mL to about 400 pg/mL, about 180
pg/mL to
about 400 pg/mL, about 190 pg/mL to about 400 pg/mL, about 200 pg/mL to about
400
pg/mL, about 150 pg/mL to about 300 pg/mL, about 160 pg/mL to about 300 pg/mL,
about
170 pg/mL to about 300 pg/mL, about 180 pg/mL to about 300 pg/mL, about 190
pg/mL to
about 300 pg/mL, or about 200 pg/mL to about 300 pg/mL.
[0139] Additionally, and alternatively, in some embodiments, the reference
level for GFAP
is from about 30 pg/mL to about 1700 pg/mL, about 40 pg/mL to about 1700
pg/mL, about
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50 pg/mL to about 1700 pg/mL, about 60 pg/mL to about 1700 pg/mL, about 70
pg/mL to
about 1700 pg/mL, about 80 pg/mL to about 1700 pg/mL, about 90 pg/mL to about
1700
pg/mL, about 100 pg/mL to about 1700 pg/mL, about 150 pg/mL to about 1700
pg/mL, about
200 pg/mL to about 1700 pg/mL, about 225 pg/mL to about 1700 pg/mL, about 250
pg/mL to
about 1700 pg/mL, about 300 pg/mL to about 1700 pg/mL, about 400 pg/mL to
about 1700
pg/mL, about 500 pg/mL to about 1700 pg/mL, about 600 pg/mL to about 1700
pg/mL, about
800 pg/mL to about 1700 pg/mL, about 900 pg/mL to about 1700 pg/mL, about 1000
pg/mL
to about 1700 pg/mL, about 30 pg/mL to about 1600 pg/mL, about 40 pg/mL to
about 1600
pg/mL, about 50 pg/mL to about 1600 pg/mL, about 60 pg/mL to about 1600 pg/mL,
about
70 pg/mL to about 1600 pg/mL, about 80 pg/mL to about 1600 pg/mL, about 90
pg/mL to
about 1600 pg/mL, about 100 pg/mL to about 1600 pg/mL, about 150 pg/mL to
about 1600
pg/mL, about 200 pg/mL to about 1600 pg/mL, about 225 pg/mL to about 1600
pg/mL, about
250 pg/mL to about 1600 pg/mL, about 300 pg/mL to about 1600 pg/mL, about 400
pg/mL to
about 1600 pg/mL, about 500 pg/mL to about 1600 pg/mL, about 600 pg/mL to
about 1600
pg/mL, about 800 pg/mL to about 1600 pg/mL, about 900 pg/mL to about 1600
pg/mL, about
1000 pg/mL to about 1600 pg/mL, about 30 pg/mL to about 1500 pg/mL, about 40
pg/mL to
about 1500 pg/mL, about 50 pg/mL to about 1500 pg/mL, about 60 pg/mL to about
1500
pg/mL, about 70 pg/mL to about 1500 pg/mL, about 80 pg/mL to about 1500 pg/mL,
about
90 pg/mL to about 1500 pg/mL, about 100 pg/mL to about 1500 pg/mL, about 150
pg/mL to
about 1500 pg/mL, about 200 pg/mL to about 1500 pg/mL, about 225 pg/mL to
about 1500
pg/mL, about 250 pg/mL to about 1500 pg/mL, about 300 pg/mL to about 1500
pg/mL, about
400 pg/mL to about 1500 pg/mL, about 500 pg/mL to about 1500 pg/mL, about 600
pg/mL to
about 1500 pg/mL, about 800 pg/mL to about 1500 pg/mL, about 900 pg/mL to
about 1500
pg/mL, about 1000 pg/mL to about 1500 pg/mL, about 30 pg/mL to about 1400
pg/mL, about
40 pg/mL to about 1400 pg/mL, about 50 pg/mL to about 1400 pg/mL, about 60
pg/mL to
about 1400 pg/mL, about 70 pg/mL to about 1400 pg/mL, about 80 pg/mL to about
1400
pg/mL, about 90 pg/mL to about 1400 pg/mL, about 100 pg/mL to about 1400
pg/mL, about
150 pg/mL to about 1400 pg/mL, about 200 pg/mL to about 1400 pg/mL, about 225
pg/mL to
about 1400 pg/mL, about 250 pg/mL to about 1400 pg/mL, about 300 pg/mL to
about 1400
pg/mL, about 400 pg/mL to about 1400 pg/mL, about 500 pg/mL to about 1400
pg/mL, about
600 pg/mL to about 1400 pg/mL, about 800 pg/mL to about 1400 pg/mL, about 900
pg/mL to
about 1400 pg/mL, about 1000 pg/mL to about 1400 pg/mL, about 30 pg/mL to
about 1300
pg/mL, about 40 pg/mL to about 1300 pg/mL, about 50 pg/mL to about 1300 pg/mL,
about
60 pg/mL to about 1300 pg/mL, about 70 pg/mL to about 1300 pg/mL, about 80
pg/mL to
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about 1300 pg/mL, about 90 pg/mL to about 1300 pg/mL, about 100 pg/mL to about
1300
pg/mL, about 150 pg/mL to about 1300 pg/mL, about 200 pg/mL to about 1300
pg/mL, about
225 pg/mL to about 1300 pg/mL, about 250 pg/mL to about 1300 pg/mL, about 300
pg/mL to
about 1300 pg/mL, about 400 pg/mL to about 1300 pg/mL, about 500 pg/mL to
about 1300
pg/mL, about 600 pg/mL to about 1300 pg/mL, about 800 pg/mL to about 1300
pg/mL, about
900 pg/mL to about 1300 pg/mL, about 1000 pg/mL to about 1300 pg/mL, about 30
pg/mL to
about 1200 pg/mL, about 40 pg/mL to about 1200 pg/mL, about 50 pg/mL to about
1200
pg/mL, about 60 pg/mL to about 1200 pg/mL, about 70 pg/mL to about 1200 pg/mL,
about
80 pg/mL to about 1200 pg/mL, about 90 pg/mL to about 1200 pg/mL, about 100
pg/mL to
about 1200 pg/mL, about 150 pg/mL to about 1200 pg/mL, about 200 pg/mL to
about 1200
pg/mL, about 225 pg/mL to about 1200 pg/mL, about 250 pg/mL to about 1200
pg/mL, about
300 pg/mL to about 1200 pg/mL, about 400 pg/mL to about 1200 pg/mL, about 500
pg/mL to
about 1200 pg/mL, about 600 pg/mL to about 1200 pg/mL, about 800 pg/mL to
about 1200
pg/mL, about 900 pg/mL to about 1200 pg/mL, or about 1000 pg/mL to about 1200
pg/mL.
[0140] In some aspects, the reference level for GFAP is from about 90 pg/mL to
about 1680
pg/mL and the reference level for UCH-L1 is from about 220 pg/mL to about 670
pg/mL. In
other aspects, the reference level for GFAP is from about 110 pg/mL to about
950 pg/mL and
the reference level for UCH-L1 is from about 160 pg/mL to about 320 pg/mL.
[0141] In some embodiments, the method further includes treating the subject,
such as a
human subject, with a traumatic brain injury treatment and/or monitoring the
subject, as
described below.
[0142] The nature of the assay employed in the methods described herein is not
critical and
the test can be any assay known in the art such as, for example, immunoassays,
protein
immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and
Western
blot analysis, or protein immunostaining, electrophoresis analysis, a protein
assay, a
competitive binding assay, a functional protein assay, or chromatography or
spectrometry
methods, such as high-performance liquid chromatography (HPLC) or liquid
chromatography¨mass spectrometry (LC/MS). Also, the assay can be employed in a
clinical
chemistry format such as would be known by one of ordinary skill in the art.
Such assays are
described in further detail herein in Sections 4-8. It is known in the art
that the values (e.g.,
reference levels, cutoffs, thresholds, specificities, sensitivities,
concentrations of calibrators
and/or controls etc.) used in an assay that employs specific sample type
(e.g., such as an
immunoassay that utilizes serum or a point-of-care device that employs whole
blood) can be
extrapolated to other assay formats using known techniques in the art, such as
assay
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standardization. For example, one way in which assay standardization can be
performed is
by applying a factor to the calibrator employed in the assay to make the
sample concentration
read higher or lower to get a slope that aligns with the comparator method.
Other methods of
standardizing results obtained on one assay to another assay are well known
and have been
described in the literature (See, for example, David Wild, Immunoassay
Handbook, 4th
edition, chapter 3.5, pages 315-322, the contents of which are herein
incorporated by
reference).
3. Treatment and Monitoring of Subjects Who Have Sustained an Injury to the
Head
101431 The subject identified in the methods described above may be treated or
monitored.
In some embodiments, the method further includes treating the subject, such as
a human
subject, with a traumatic brain injury treatment, such as any treatments known
in the art. For
example, treatment of traumatic brain injury can take a variety of forms
depending on the
severity of the injury to the head. For example, for subjects suffering from
mild TBI, the
treatment may include one or more of rest, abstaining from physical
activities, such as sports,
avoiding light or wearing sunglasses when out in the light, medication for
relief of a headache
or migraine, anti-nausea medication, etc. Treatment for patients suffering
from moderate,
severe or moderate to severe TBI might include administration of one or more
appropriate
medications (such as, for example, diuretics, anti-convulsant medications,
medications to
sedate and put an individual in a drug-induced coma, or other pharmaceutical
or
biopharmaceutical medications (either known or developed in the future for
treatment of
TBI), one or more surgical procedures (such as, for example, removal of a
hematoma,
repairing a skull fracture, decompressive craniectomy, etc.), protecting the
airway, and one or
more therapies (such as, for example one or more rehabilitation, cognitive
behavioral therapy,
anger management, counseling psychology, etc.). In some embodiments, the
method further
includes monitoring the subject, such as a human subject. In some embodiments,
a subject
may be monitored with CT scan or MRI procedure.
4. Methods for Measuring the Level of UCH-L1
101441 In the methods described above, UCH-L1 levels can be measured by any
means,
such as antibody dependent methods, such as immunoassays, protein
immunoprecipitation,
immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis,
protein
immunostaining, electrophoresis analysis, a protein assay, a competitive
binding assay, a
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functional protein assay, or chromatography or spectrometry methods, such as
high-
performance liquid chromatography (HPLC) or liquid chromatography¨mass
spectrometry
(LC/MS). Also, the assay can be employed in clinical chemistry format such as
would be
known by one skilled in the art.
101451 In some embodiments, measuring the level of UCH-L1 includes contacting
the
sample with a first specific binding member and second specific binding
member. In some
embodiments the first specific binding member is a capture antibody and the
second specific
binding member is a detection antibody. In some embodiments, measuring the
level of UCH-
Li includes contacting the sample, either simultaneously or sequentially, in
any order: (1) a
capture antibody (e.g., UCH-Li-capture antibody), which binds to an epitope on
UCH-L1 or
UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., UCH-
Li-
capture antibody-UCH-Li antigen complex), and (2) a detection antibody (e.g.,
UCH-L1-
detection antibody), which includes a detectable label and binds to an epitope
on UCH-L1
that is not bound by the capture antibody, to form a UCH-L1 antigen-detection
antibody
complex (e.g., UCH-L1 antigen-UCH-Li-detection antibody complex), such that a
capture
antibody-UCH-L1 antigen-detection antibody complex (e.g., UCH-L I-capture
antibody-
UCH-L1 antigen-UCH-L I-detection antibody complex) is formed, and measuring
the amount
or concentration of UCH-L1 in the sample based on the signal generated by the
detectable
label in the capture antibody-UCH-L1 antigen-detection antibody complex.
101461 In some embodiments, the first specific binding member is immobilized
on a solid
support. In some embodiments, the second specific binding member is
immobilized on a
solid support. In some embodiments, the first specific binding member is a UCH-
L1
antibody as described below.
101471 In some embodiments, the sample is diluted or undiluted. The sample can
be from
about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to
about 23
microliters, about 1 to about 22 microliters, about 1 to about 21 microliters,
about 1 to about
20 microliters, about 1 to about 18 microliters, about 1 to about 17
microliters, about 1 to
about 16 microliters, about 15 microliters or about 1 microliter, about 2
microliters, about 3
microliters, about 4 microliters, about 5 microliters, about 6 microliters,
about 7 microliters,
about 8 microliters, about 9 microliters, about 10 microliters, about 11
microliters, about 12
microliters, about 13 microliters, about 14 microliters, about 15 microliters,
about 16
microliters, about 17 microliters, about 18 microliters, about 19 microliters,
about 20
microliters, about 21 microliters, about 22 microliters, about 23 microliters,
about 24
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microliters or about 25 microliters. In some embodiments, the sample is from
about 1 to
about 150 microliters or less or from about 1 to about 25 microliters or less.
[0148] Some instruments (such as, for example the Abbott Laboratories
instrument
ARCHITECT , and other core laboratory instruments) other than a point-of-care
device may
be capable of measuring levels of UCH-L1 in a sample higher or greater than
25,000 pg/mL.
[0149] Other methods of detection include the use of or can be adapted for use
on a
nanopore device or nanowell device. Examples of nanopore devices are described
in
International Patent Publication No. WO 2016/161402, which is hereby
incorporated by
reference in its entirety. Examples of nanowell device are described in
International Patent
Publication No. WO 2016/161400, which is hereby incorporated by reference in
its entirety
5. UCH-L1 Antibodies
[0150] The methods described herein may use an isolated antibody that
specifically binds to
ubiquitin carboxy-terminal hydrolase Li ("UCH-L1") (or fragments thereof),
referred to as
"UCH-L1 antibody." The UCH-L1 antibodies can be used to assess the UCH-L1
status as a
measure of traumatic brain injury, detect the presence of UCH-L1 in a sample,
quantify the
amount of UCH-L1 present in a sample, or detect the presence of and quantify
the amount of
UCH-L1 in a sample.
a. Ubiquitin Carboxy-Terminal Hydrolase Li (UCH-L1)
[0151] Ubiquitin carboxy-terminal hydrolase Li ("UCH-L1"), which is also known
as
"ubiquitin C-terminal hydrolase," is a deubiquitinating enzyme. UCH-L1 is a
member of a
gene family whose products hydrolyze small C-terminal adducts of ubiquitin to
generate the
ubiquitin monomer. Expression of UCH-L1 is highly specific to neurons and to
cells of the
diffuse neuroendocrine system and their tumors. It is abundantly present in
all neurons
(accounts for 1-2% of total brain protein), expressed specifically in neurons
and testis/ovary.
The catalytic triad of UCH-L1 contains a cysteine at position 90, an aspartate
at position 176,
and a histidine at position 161 that are responsible for its hydrolase
activity.
[0152] Human UCH-L1 may have the following amino acid sequence:
[0153] MQLKPMEINPEMLNKVLSRLGVAGQWREVDVLGLEEESLGSVPAPACALLL
LFPLTAQHENFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGF
EDGSVLKQFLSETEKMSPEDRAKCFEKNEAIQAAHDAVAQEGQCRVDDKVNFHFIL
FNNVDGHLYELDGRMPFPVNHGASSEDTLLKDAAKVCREFTEREQGEVRFSAVALC
KAA (SEQ ID NO: 1).
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[0154] The human UCH-L1 may be a fragment or variant of SEQ ID NO: 1. The
fragment
of UCH-L1 may be between 5 and 225 amino acids, between 10 and 225 amino
acids,
between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and
225
amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids,
between
100 and 175 amino acids, or between 175 and 225 amino acids in length. The
fragment may
comprise a contiguous number of amino acids from SEQ ID NO: 1.
b. UCH-Li-Recognizing Antibody
[0155] The antibody is an antibody that binds to UCH-L1, a fragment thereof,
an epitope of
UCH-L1, or a variant thereof. The antibody may be a fragment of the anti-UCH-
L1 antibody
or a variant or a derivative thereof. The antibody may be a polyclonal or
monoclonal
antibody. The antibody may be a chimeric antibody, a single chain antibody, an
affinity
matured antibody, a human antibody, a humanized antibody, a fully human
antibody or an
antibody fragment, such as a Fab fragment, or a mixture thereof. Antibody
fragments or
derivatives may comprise F(ab')-,, Fv or scFv fragments. The antibody
derivatives can be
produced by peptidomimetics. Further, techniques described for the production
of single
chain antibodies can be adapted to produce single chain antibodies.
[0156] The anti-UCH-L1 antibodies may be a chimeric anti-UCH-L1 or humanized
anti-
UCH-L1 antibody. In one embodiment, both the humanized antibody and chimeric
antibody
are monovalent. In one embodiment, both the humanized antibody and chimeric
antibody
comprise a single Fab region linked to an Fc region.
[0157] Human antibodies may be derived from phage-display technology or from
transgenic mice that express human immunoglobulin genes. The human antibody
may be
generated as a result of a human in vivo immune response and isolated. See,
for example,
Funaro et al., BMC Biotechnology, 2008(8):85. Therefore, the antibody may be a
product of
the human and not animal repertoire. Because it is of human origin, the risks
of reactivity
against self-antigens may be minimized. Alternatively, standard yeast display
libraries and
display technologies may be used to select and isolate human anti-UCH-L1
antibodies. For
example, libraries of naive human single chain variable fragments (scFv) may
be used to
select human anti-UCH-L1 antibodies. Transgenic animals may be used to express
human
antibodies.
[0158] Humanized antibodies may be antibody molecules from non-human species
antibody that binds the desired antigen having one or more complementarity
determining
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regions (CDRs) from the non-human species and framework regions from a human
immunoglobulin molecule_
[0159] The antibody is distinguishable from known antibodies in that it
possesses different
biological function(s) than those known in the art.
(1) Epitope
[0160] The antibody may immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a
fragment
thereof, or a variant thereof. The antibody may immunospecifically recognize
and bind at
least three amino acids, at least four amino acids, at least five amino acids,
at least six amino
acids, at least seven amino acids, at least eight amino acids, at least nine
amino acids, or at
least ten amino acids within an epitope region. The antibody may
immunospecifically
recognize and bind to an epitope that has at least three contiguous amino
acids, at least four
contiguous amino acids, at least five contiguous amino acids, at least six
contiguous amino
acids, at least seven contiguous amino acids, at least eight contiguous amino
acids, at least
nine contiguous amino acids, or at least ten contiguous amino acids of an
epitope region.
c. Antibody Preparation/Production
[0161] Antibodies may be prepared by any of a variety of techniques, including
those well
known to those skilled in the art. In general, antibodies can be produced by
cell culture
techniques, including the generation of monoclonal antibodies via conventional
techniques,
or via transfection of antibody genes, heavy chains, and/or light chains into
suitable bacterial
or mammalian cell hosts, to allow for the production of antibodies, wherein
the antibodies
may be recombinant. The various forms of the term "transfection" are intended
to encompass
a wide variety of techniques commonly used for the introduction of exogenous
DNA into a
prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation,
DEAE-dextran transfection and the like. Although it is possible to express the
antibodies in
either prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is
preferable, and most preferable in mammalian host cells, because such
eukaryotic cells (and
in particular mammalian cells) are more likely than prokaryotic cells to
assemble and secrete
a properly folded and immunologically active antibody.
[0162] Exemplary mammalian host cells for expressing the recombinant
antibodies include
Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in
Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), used with a DHFR
selectable
marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621
(1982), NSO
myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors
encoding
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antibody genes are introduced into mammalian host cells, the antibodies are
produced by
culturing the host cells for a period of time sufficient to allow for
expression of the antibody
in the host cells or, more preferably, secretion of the antibody into the
culture medium in
which the host cells are grown. Antibodies can be recovered from the culture
medium using
standard protein purification methods.
[0163] Host cells can also be used to produce functional antibody fragments,
such as Fab
fragments or scFv molecules. It will be understood that variations on the
above procedure
may be performed. For example, it may be desirable to transfect a host cell
with DNA
encoding functional fragments of either the light chain and/or the heavy chain
of an antibody.
Recombinant DNA technology may also be used to remove some, or all, of the DNA
encoding either or both of the light and heavy chains that is not necessary
for binding to the
antigens of interest. The molecules expressed from such truncated DNA
molecules are also
encompassed by the antibodies. In addition, bifunctional antibodies may be
produced in
which one heavy and one light chain are an antibody (i.e., binds human UCH-Li)
and the
other heavy and light chain are specific for an antigen other than human UCH-
Li by
crosslinking an antibody to a second antibody by standard chemical
crosslinking methods.
101641 In a preferred system for recombinant expression of an antibody, or
antigen-binding
portion thereof, a recombinant expression vector encoding both the antibody
heavy chain and
the antibody light chain is introduced into dhfr-CHO cells by calcium
phosphate-mediated
transfection. Within the recombinant expression vector, the antibody heavy and
light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory
elements to
drive high levels of transcription of the genes. The recombinant expression
vector also
carries a DHFR gene, which allows for selection of CHO cells that have been
transfected
with the vector using methotrexate selection/amplification. The selected
transformant host
cells are cultured to allow for expression of the antibody heavy and light
chains and intact
antibody is recovered from the culture medium. Standard molecular biology
techniques are
used to prepare the recombinant expression vector, transfect the host cells,
select for
transformants, culture the host cells, and recover the antibody from the
culture medium. Still
further, the method of synthesizing a recombinant antibody may be by culturing
a host cell in
a suitable culture medium until a recombinant antibody is synthesized. The
method can
further comprise isolating the recombinant antibody from the culture medium.
[0165] Methods of preparing monoclonal antibodies involve the preparation of
immortal
cell lines capable of producing antibodies having the desired specificity.
Such cell lines may
be produced from spleen cells obtained from an immunized animal. The animal
may be
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immunized with UCH-L1 or a fragment and/or variant thereof. The peptide used
to
immunize the animal may comprise amino acids encoding human Fc, for example
the
fragment crystallizable region or tail region of human antibody. The spleen
cells may then be
immortalized by, for example, fusion with a myeloma cell fusion partner. A
variety of fusion
techniques may be employed. For example, the spleen cells and myeloma cells
may be
combined with a nonionic detergent for a few minutes and then plated at low
density on a
selective medium that supports that growth of hybrid cells, but not myeloma
cells. One such
technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another
technique
includes electrofusion. After a sufficient time, usually about 1 to 2 weeks,
colonies of
hybrids are observed. Single colonies are selected and their culture
supernatants tested for
binding activity against the polypeptide. Hybridomas having high reactivity
and specificity
may be used.
[0166] Monoclonal antibodies may be isolated from the supernatants of growing
hybridoma
colonies. In addition, various techniques may be employed to enhance the
yield, such as
injection of the hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host,
such as a mouse. Monoclonal antibodies may then be harvested from the ascites
fluid or the
blood. Contaminants may be removed from the antibodies by conventional
techniques, such
as chromatography, gel filtration, precipitation, and extraction. Affinity
chromatography is
an example of a method that can be used in a process to purify the antibodies.
[0167] The proteolytic enzyme papain preferentially cleaves IgG molecules to
yield several
fragments, two of which (the F(ab) fragments) each comprise a covalent
heterodimer that
includes an intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to
provide several fragments, including the F(ab'), fragment, which comprises
both antigen-
binding sites.
[0168] The Fv fragment can be produced by preferential proteolytic cleavage of
an IgM,
and on rare occasions IgG or IgA immunoglobulin molecules. The Fv fragment may
be
derived using recombinant techniques. The Fv fragment includes a non-covalent
VH::VL
heterodimer including an antigen-binding site that retains much of the antigen
recognition
and binding capabilities of the native antibody molecule.
[0169] The antibody, antibody fragment, or derivative may comprise a heavy
chain and a
light chain complementarity determining region ("CDR") set, respectively
interposed
between a heavy chain and a light chain framework ("FR") set which provide
support to the
CDRs and define the spatial relationship of the CDRs relative to each other.
The CDR set
may contain three hypervariable regions of a heavy or light chain V region.
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[0170] Other suitable methods of producing or isolating antibodies of the
requisite
specificity can be used, including, but not limited to, methods that select
recombinant
antibody from a peptide or protein library (e.g., but not limited to, a
bacteriophage, ribosome,
oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g., as
available from
various commercial vendors such as Cambridge Antibody Technologies
(Cambridgeshire,
UK), Morph Sys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK)
BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos.
4,704,692;
5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative
methods rely
upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1997)
Microbiol.
Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118;
Eren et al.
(1998) Immunol. 93:154-161) that are capable of producing a repertoire of
human antibodies,
as known in the art and/or as described herein. Such techniques, include, but
are not limited
to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sc!. USA, 94:4937-
4942; Hanes et
al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody
producing
technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent
No.
5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996)
Proc. Natl.
Acad. Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell
etal. (1990)
Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al.
(1995) J. Imm.
Meth. 182:155-163; Kenny et al. (1995) Bio/Technol. 13:787-790); B-cell
selection
(Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).
[0171] An affinity matured antibody may be produced by any one of a number of
procedures that are known in the art. For example, see Marks et al.,
BioTechnology, 10: 779-
783 (1992) describes affinity maturation by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by Barbas et al.,
Proc. Nat.
Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155
(1995); Yelton et
al., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7):
3310-3319
(1995); Hawkins el al, I Mol. Biol., 226: 889-896 (1992). Selective mutation
at selective
mutagenesis positions and at contact or hypermutation positions with an
activity enhancing
amino acid residue is described in U.S. Patent No. 6,914,128 BI.
[0172] Antibody variants can also be prepared using delivering a
polynucleotide encoding
an antibody to a suitable host such as to provide transgenic animals or
mammals, such as
goats, cows, horses, sheep, and the like, that produce such antibodies in
their milk. These
methods are known in the art and are described for example in U.S. Patent Nos.
5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.
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[0173] Antibody variants also can be prepared by delivering a polynucleotide
to provide
transgenic plants and cultured plant cells (e.g., but not limited to tobacco,
maize, and
duckweed) that produce such antibodies, specified portions or variants in the
plant parts or in
cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.
Microbiol. Immunol.
240:95-118 and references cited therein, describe the production of transgenic
tobacco leaves
expressing large amounts of recombinant proteins, e.g., using an inducible
promoter.
Transgenic maize have been used to express mammalian proteins at commercial
production
levels, with biological activities equivalent to those produced in other
recombinant systems or
purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol.
(1999) 464:127-
147 and references cited therein. Antibody variants have also been produced in
large
amounts from transgenic plant seeds including antibody fragments, such as
single chain
antibodies (scFvs), including tobacco seeds and potato tubers. See, e.g.,
Conrad et al. (1998)
Plant Mol. Biol. 38:101-109 and reference cited therein. Thus, antibodies can
also be
produced using transgenic plants, according to known methods.
[0174] Antibody derivatives can be produced, for example, by adding exogenous
sequences
to modify immunogenicity or reduce, enhance or modify binding, affinity, on-
rate, off-rate,
avidity, specificity, half-life, or any other suitable characteristic.
Generally, part or all of the
non-human or human CDR sequences are maintained while the non-human sequences
of the
variable and constant regions are replaced with human or other amino acids.
[0175] Small antibody fragments may be diabodies having two antigen-binding
sites,
wherein fragments comprise a heavy chain variable domain (VH) connected to a
light chain
variable domain (VL) in the same polypeptide chain (VH VL). See for example,
EP 404,097;
WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-
6448. By
using a linker that is too short to allow pairing between the two domains on
the same chain,
the domains are forced to pair with the complementary domains of another chain
and create
two antigen-binding sites. See also, U.S. Patent No. 6,632,926 to Chen et al.
which is hereby
incorporated by reference in its entirety and discloses antibody variants that
have one or more
amino acids inserted into a hypervariable region of the parent antibody and a
binding affinity
for a target antigen which is at least about two fold stronger than the
binding affinity of the
parent antibody for the antigen.
[0176] The antibody may be a linear antibody. The procedure for making a
linear antibody
is known in the art and described in Zapata et al., (1995) Protein Eng.
8(10):1057-1062.
Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-
CH1) which
form a pair of antigen binding regions. Linear antibodies can be bispecific or
monospecific.
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[0177] The antibodies may be recovered and purified from recombinant cell
cultures by
known methods including, but not limited to, protein A purification, ammonium
sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be used for purification.
[0178] It may be useful to detectably label the antibody. Methods for
conjugating
antibodies to these agents are known in the art. For the purpose of
illustration only,
antibodies can be labeled with a detectable moiety such as a radioactive atom,
a
chromophore, a fluorophore, or the like. Such labeled antibodies can be used
for diagnostic
techniques, either in vivo, or in an isolated test sample. They can be linked
to a cytokine, to a
ligand, to another antibody. Suitable agents for coupling to antibodies to
achieve an anti-
tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor
Necrosis Factor
(TNF); photosensitizers, for use in photodynamic therapy, including aluminum
(III)
phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine;
radionuclides, such as
iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),
technetium-
99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such
as
doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,
neocarzinostatin, and
carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin,
pseudomonas
exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A
(deglycosylated ricin A and
native ricin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja atra),
and gelonin (a
plant toxin); ribosome inactivating proteins from plants, bacteria and fungi,
such as
restrictocin (a ribosome inactivating protein produced by Aspergillus
restrictus), saporin (a
ribosome inactivating protein from Saponaria otTicinalis), and RNase; tyrosine
kinase
inhibitors; 1y207702 (a difluorinated purine nucleoside); liposomes containing
anti cystic
agents (e.g., antisense oligonucleotides, plasmids which encode for toxins,
methotrexate,
etc.); and other antibodies or antibody fragments, such as F(ab).
[0179] Antibody production via the use of hybridoma technology, the selected
lymphocyte
antibody method (SLAM), transgenic animals, and recombinant antibody libraries
is
described in more detail below.
(1) Anti-UCH-L1 Monoclonal Antibodies Using Hybridoma Technology
[0180] Monoclonal antibodies can be prepared using a wide variety of
techniques known in
the art including the use of hybridoma, recombinant, and phage display
technologies, or a
combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
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techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, second edition, (Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, 1988); Hammerling, et al., In Monoclonal Antibodies and T-
Cell
Hybridomas, (Elsevier, N.Y., 1981). It is also noted that the term "monoclonal
antibody" as
used herein is not limited to antibodies produced through hybridoma
technology. The term
"monoclonal antibody" refers to an antibody that is derived from a single
clone, including
any eukaryotic, prokaryotic, or phage clone, and not the method by which it is
produced.
[0181] Methods of generating monoclonal antibodies as well as antibodies
produced by the
method may comprise culturing a hybridoma cell secreting an antibody of the
invention
wherein, preferably, the hybridoma is generated by fusing splenocytes isolated
from an
animal, e.g., a rat or a mouse, immunized with UCH-L1 with myeloma cells and
then
screening the hybridomas resulting from the fusion for hybridoma clones that
secrete an
antibody able to bind a polypeptide of the invention. Briefly, rats can be
immunized with a
UCH-L1 antigen. In a preferred embodiment, the UCH-L1 antigen is administered
with an
adjuvant to stimulate the immune response. Such adjuvants include complete or
incomplete
Freund's adjuvant, RIB I (muramyl dipeptides) or ISCOM (immunostimulating
complexes).
Such adjuvants may protect the polypeptide from rapid dispersal by
sequestering it in a local
deposit, or they may contain substances that stimulate the host to secrete
factors that are
chemotactic for macrophages and other components of the immune system.
Preferably, if a
polypeptide is being administered, the immunization schedule will involve two
or more
administrations of the polypeptide, spread out over several weeks; however, a
single
administration of the polypeptide may also be used.
[0182] After immunization of an animal with a UCH-L1 antigen, antibodies
and/or
antibody-producing cells may be obtained from the animal. An anti-UCH-L1
antibody-
containing serum is obtained from the animal by bleeding or sacrificing the
animal. The
serum may be used as it is obtained from the animal, an immunoglobulin
fraction may be
obtained from the serum, or the anti-UCH-L1 antibodies may be purified from
the serum.
Serum or immunoglobulins obtained in this manner are polyclonal, thus having a
heterogeneous array of properties.
[0183] Once an immune response is detected, e.g., antibodies specific for the
antigen UCH-
Li are detected in the rat serum, the rat spleen is harvested and splenocytes
isolated. The
splenocytes are then fused by well-known techniques to any suitable myeloma
cells, for
example, cells from cell line SP20 available from the American Type Culture
Collection
(ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limited
dilution. The
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hybridoma clones are then assayed by methods known in the art for cells that
secrete
antibodies capable of binding UCH-L1 Ascites fluid, which generally contains
high levels
of antibodies, can be generated by immunizing rats with positive hybridoma
clones.
[0184] In another embodiment, antibody-producing immortalized hybridomas may
be
prepared from the immunized animal. After immunization, the animal is
sacrificed, and the
splenic B cells are fused to immortalized myeloma cells as is well known in
the art. See, e.g.,
Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not
secrete
immunoglobulin polypeptides (a non-secretory cell line). After fusion and
antibiotic
selection, the hybridomas are screened using UCH-L1, or a portion thereof, or
a cell
expressing UCH-L1. In a preferred embodiment, the initial screening is
performed using an
enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA),
preferably an
ELISA. An example of ELISA screening is provided in PCT Publication No. WO
00/37504.
[0185] Anti-UCH-L1 antibody-producing hybridomas are selected, cloned, and
further
screened for desirable characteristics, including robust hybridoma growth,
high antibody
production, and desirable antibody characteristics. Hybridomas may be cultured
and
expanded in vivo in syngeneic animals, in animals that lack an immune system,
e.g., nude
mice, or in cell culture in vitro. Methods of selecting, cloning and expanding
hybridomas are
well known to those of ordinary skill in the art.
[0186] In a preferred embodiment, hybridomas are rat hybridomas. In another
embodiment,
hybridomas are produced in a non-human, non-rat species such as mice, sheep,
pigs, goats,
cattle, or horses. In yet another preferred embodiment, the hybridomas are
human
hybridomas, in which a human non-secretory myeloma is fused with a human cell
expressing
an anti-UCH-L1 antibody.
[0187] Antibody fragments that recognize specific epitopes may be generated by
known
techniques. For example, Fab and F(ab')2 fragments of the invention may be
produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain
(to
produce two identical Fab fragments) or pepsin (to produce an F(ab')/
fragment). A F(ab1)2
fragment of an IgG molecule retains the two antigen-binding sites of the
larger ("parent") IgG
molecule, including both light chains (containing the variable light chain and
constant light
chain regions), the CH1 domains of the heavy chains, and a disulfide-forming
hinge region of
the parent IgG molecule. Accordingly, an F(ab')1 fragment is still capable of
crosslinking
antigen molecules like the parent IgG molecule.
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(2) Anti-UCH-L1 Monoclonal Antibodies Using SLAM
101881 In another aspect of the invention, recombinant antibodies are
generated from single,
isolated lymphocytes using a procedure referred to in the art as the selected
lymphocyte
antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT
Publication No.
WO 92/02551; and Babcook el al., Proc. Nall. Acad. Sci. USA, 93: 7843-7848
(1996). In this
method, single cells secreting antibodies of interest, e.g., lymphocytes
derived from any one
of the immunized animals are screened using an antigen-specific hemolytic
plaque assay,
wherein the antigen UCH-L1, a subunit of UCH-L1, or a fragment thereof, is
coupled to
sheep red blood cells using a linker, such as biotin, and used to identify
single cells that
secrete antibodies with specificity for UCH-L1. Following identification of
antibody-
secreting cells of interest, heavy- and light-chain variable region cDNAs are
rescued from the
cells by reverse transcriptase-PCR (RT-PCR) and these variable regions can
then be
expressed, in the context of appropriate immunoglobulin constant regions
(e.g., human
constant regions), in mammalian host cells, such as COS or CHO cells. The host
cells
transfected with the amplified immunoglobulin sequences, derived from in vivo
selected
lymphocytes, can then undergo further analysis and selection in vitro, for
example, by
panning the transfected cells to isolate cells expressing antibodies to UCH-
L1. The amplified
immunoglobulin sequences further can be manipulated in vitro, such as by in
vitro affinity
maturation method. See, for example, PCT Publication No. WO 97/29131 and PCT
Publication No. WO 00/56772.
(3) Anti-UCH-L1 Monoclonal Antibodies Using Transgenic Animals
[0189] In another embodiment of the invention, antibodies are produced by
immunizing a
non-human animal comprising some, or all, of the human immunoglobulin locus
with a
UCH-L1 antigen. In an embodiment, the non-human animal is a XENOMOUSEO
transgenic
mouse, an engineered mouse strain that comprises large fragments of the human
immunoglobulin loci and is deficient in mouse antibody production. See. e.g.,
Green et al.,
Nature Genetics, 7: 13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598;
5,985,615;
5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT
Publication Nos.
WO 91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO 98/24893;
WO 98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The
XENOMOUSEO transgenic mouse produces an adult-like human repertoire of fully
human
antibodies and generates antigen-specific human monoclonal antibodies. The
XENOMOUSEO transgenic mouse contains approximately 80% of the human antibody
repertoire through introduction of megabase sized, germline configuration YAC
fragments of
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the human heavy chain loci and x light chain loci. See Mendez et al., Nature
Genetics, 15:
146-156 (1997), Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the
disclosures of
which are hereby incorporated by reference.
(4) Anti-UCH-L1 Monoclonal Antibodies Using Recombinant Antibody
Libraries
[0190] In vitro methods also can be used to make the antibodies of the
invention, wherein
an antibody library is screened to identify an antibody having the desired UCH-
L1 -binding
specificity. Methods for such screening of recombinant antibody libraries are
well known in
the art and include methods described in, for example, U.S. Patent No.
5,223,409 (Ladner et
al.); PCT Publication No. WO 92/18619 (Kang et al.); PCT Publication No. WO
91/17271
(Dower et al.); PCT Publication No. WO 92/20791 (Winter et al.); PCT
Publication No. WO
92/15679 (Markland et al.); PCT Publication No. WO 93/01288 (Breitling et
al.); PCT
Publication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO
92/09690
(Garrard et al.); Fuchs et al., Bio/Technology, 9: 1369-1372 (1991); Hay et
al., Hum.
Antibod. Hybridomas, 3: 81-85 (1992); Huse et al.õScience, 246: 1275-1281
(1989);
McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12:
725-734
(1993); Hawkins et al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al.,
Nature, 352: 624-
628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992);
Garrard et al.,
Bin/Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res,, 19:
4133-4137
(1991); Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); U.S.
Patent
Application Publication No. 2003/0186374; and PCT Publication No. WO 97/29131,
the
contents of each of which are incorporated herein by reference.
[0191] The recombinant antibody library may be from a subject immunized with
UCH-L1,
or a portion of UCH-L1. Alternatively, the recombinant antibody library may be
from a naive
subject, i.e., one who has not been immunized with UCH-L1, such as a human
antibody
library from a human subject who has not been immunized with human UCH-L1.
Antibodies
of the invention are selected by screening the recombinant antibody library
with the peptide
comprising human UCH-L1 to thereby select those antibodies that recognize UCH-
Li.
Methods for conducting such screening and selection are well known in the art,
such as
described in the references in the preceding paragraph. To select antibodies
of the invention
having particular binding affinities for UCH-L1, such as those that dissociate
from human
UCH-L1 with a particular Koff rate constant, the art-known method of surface
plasmon
resonance can be used to select antibodies having the desired Koff rate
constant. To select
antibodies of the invention having a particular neutralizing activity for hUCH-
L1, such as
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those with a particular IC50, standard methods known in the art for assessing
the inhibition of
UCH-L1 activity may be used.
[0192] In one aspect, the invention pertains to an isolated antibody, or an
antigen-binding
portion thereof, that binds human UCH-Li. Preferably, the antibody is a
neutralizing
antibody. In various embodiments, the antibody is a recombinant antibody or a
monoclonal
antibody.
[0193] For example, antibodies can also be generated using various phage
display methods
known in the art. In phage display methods, functional antibody domains are
displayed on the
surface of phage particles which carry the polynucleotide sequences encoding
them. Such
phage can be utilized to display antigen-binding domains expressed from a
repertoire or
combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding
domain that binds the antigen of interest can be selected or identified with
antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or bead. Phage
used in these
methods are typically filamentous phage including fd and M13 binding domains
expressed
from phage with Fab, Fv, or disulfide stabilized Fv antibody domains
recombinantly fused to
either the phage gene III or gene VIII protein. Examples of phage display
methods that can be
used to make the antibodies include those disclosed in Brinkmann et al., J.
Immunol.
Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186
(1995);
Kettleborough et al., Eur. J. Immunol., 24: 952-958 (1994); Persic et al.,
Gene, 187: 9-18
(1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT
Publication No.
WO 92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos.
5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.
[0194] As described in the above references, after phage selection, the
antibody coding
regions from the phage can be isolated and used to generate whole antibodies
including
human antibodies or any other desired antigen binding fragment, and expressed
in any
desired host, including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as
described in detail below. For example, techniques to recombinantly produce
Fab, Fab', and
F(ab')2 fragments can also be employed using methods known in the art such as
those
disclosed in PCT publication No. WO 92/22324; Mullinax et al., BioTechniques,
12(6): 864-
869 (1992); Sawai etal., Am. J. Reprod. Immunol., 34: 26-34 (1995); and Better
et al.,
Science, 240: 1041-1043 (1988). Examples of techniques which can be used to
produce
single-chain Fvs and antibodies include those described in U.S. Patent Nos.
4,946,778 and
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5,258,498; Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et
al., Proc. Natl.
Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science, 240: 1038-
1041 (1988).
101951 Alternative to screening of recombinant antibody libraries by phage
display, other
methodologies known in the art for screening large combinatorial libraries can
be applied to
the identification of antibodies of the invention. One type of alternative
expression system is
one in which the recombinant antibody library is expressed as RNA-protein
fusions, as
described in PCT Publication No. WO 98/31700 (Szostak and Roberts), and in
Roberts and
Szostak, Proc. Natl. Acad. Set. USA, 94: 12297-12302 (1997). In this system, a
covalent
fusion is created between an mRNA and the peptide or protein that it encodes
by in vitro
translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor
antibiotic, at their
3' end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a
combinatorial library) based on the properties of the encoded peptide or
protein, e.g.,
antibody, or portion thereof, such as binding of the antibody, or portion
thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or portions
thereof,
recovered from screening of such libraries can be expressed by recombinant
means as
described above (e.g., in mammalian host cells) and, moreover, can be
subjected to further
affinity maturation by either additional rounds of screening of mRNA-peptide
fusions in
which mutations have been introduced into the originally selected sequence(s),
or by other
methods for affinity maturation in vitro of recombinant antibodies, as
described above. A
preferred example of this methodology is PROfusion display technology.
101961 In another approach, the antibodies can also be generated using yeast
display
methods known in the art. In yeast display methods, genetic methods are used
to tether
antibody domains to the yeast cell wall and display them on the surface of
yeast. Such yeast
can be utilized to display antigen-binding domains expressed from a repertoire
or
combinatorial antibody library (e.g., human or murine). Examples of yeast
display methods
that can be used to make the antibodies include those disclosed in U.S. Patent
No. 6,699,658
(Wittrup et al.) incorporated herein by reference.
d. Production of Recombinant UCH-L1 Antibodies
101971 Antibodies may be produced by any of a number of techniques known in
the art.
For example, expression from host cells, wherein expression vector(s) encoding
the heavy
and light chains is (are) transfected into a host cell by standard techniques.
The various forms
of the term "transfection" are intended to encompass a wide variety of
techniques commonly
used for the introduction of exogenous DNA into a prokaryotic or eukaryotic
host cell, e.g.,
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electroporation, calcium-phosphate precipitation. DEAE-dextran transfection,
and the like.
Although it is possible to express the antibodies of the invention in either
prokaryotic or
eukaryotic host cells, expression of antibodies in eukaryotic cells is
preferable, and most
preferable in mammalian host cells, because such eukaryotic cells (and in
particular
mammalian cells) are more likely than prokaryotic cells to assemble and
secrete a properly
folded and immunologically active antibody.
101981 Exemplary mammalian host cells for expressing the recombinant
antibodies of the
invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,
described
in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980), used
with a DHFR
selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol.,
159: 601-621
(1982), NSO myeloma cells, COS cells, and SP2 cells. When recombinant
expression vectors
encoding antibody genes are introduced into mammalian host cells, the
antibodies are
produced by culturing the host cells for a period of time sufficient to allow
for expression of
the antibody in the host cells or, more preferably, secretion of the antibody
into the culture
medium in which the host cells are grown. Antibodies can be recovered from the
culture
medium using standard protein purification methods.
101991 Host cells can also be used to produce functional antibody fragments,
such as Fab
fragments or scFv molecules. It will be understood that variations on the
above procedure
may be performed. For example, it may be desirable to transfect a host cell
with DNA
encoding functional fragments of either the light chain and/or the heavy chain
of an antibody
of this invention. Recombinant DNA technology may also be used to remove some,
or all, of
the DNA encoding either or both of the light and heavy chains that is not
necessary for
binding to the antigens of interest. The molecules expressed from such
truncated DNA
molecules are also encompassed by the antibodies of the invention. In
addition, bifunctional
antibodies may be produced in which one heavy and one light chain are an
antibody of the
invention (i.e., binds human UCH-L1) and the other heavy and light chain are
specific for an
antigen other than human UCH-L1 by crosslinking an antibody of the invention
to a second
antibody by standard chemical crosslinking methods.
102001 In a preferred system for recombinant expression of an antibody, or
antigen-binding
portion thereof, of the invention, a recombinant expression vector encoding
both the antibody
heavy chain and the antibody light chain is introduced into dhfr-CHO cells by
calcium
phosphate-mediated transfection. Within the recombinant expression vector, the
antibody
heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP
promoter
regulatory elements to drive high levels of transcription of the genes. The
recombinant
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expression vector also carries a DHFR gene, which allows for selection of CHO
cells that
have been transfected with the vector using methotrex ate
selection/amplification. The
selected transformant host cells are cultured to allow for expression of the
antibody heavy
and light chains and intact antibody is recovered from the culture medium.
Standard
molecular biology techniques are used to prepare the recombinant expression
vector, transfect
the host cells, select for transformants, culture the host cells, and recover
the antibody from
the culture medium. Still further, the invention provides a method of
synthesizing a
recombinant antibody of the invention by culturing a host cell of the
invention in a suitable
culture medium until a recombinant antibody of the invention is synthesized.
The method
can further comprise isolating the recombinant antibody from the culture
medium.
(1) Humanized Antibody
102011 The humanized antibody may be an antibody or a variant, derivative,
analog or
portion thereof which immunospecifically binds to an antigen of interest and
which
comprises a framework (FR) region having substantially the amino acid sequence
of a human
antibody and a complementary determining region (CDR) having substantially the
amino
acid sequence of a non-human antibody. The humanized antibody may be from a
non-human
species antibody that binds the desired antigen having one or more
complementarity
determining regions (CDRs) from the non-human species and framework regions
from a
human immunoglobulin molecule.
102021 As used herein, the term "substantially" in the context of a CDR refers
to a CDR
having an amino acid sequence at least 90%, at least 95%, at least 98% or at
least 99%
identical to the amino acid sequence of a non-human antibody CDR. A humanized
antibody
comprises substantially all of at least one, and typically two, variable
domains (Fab, Fab',
F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions
correspond to those of
a non-human immunoglobulin (i.e., donor antibody) and all or substantially all
of the
framework regions are those of a human immunoglobulin consensus sequence.
According to
one aspect, a humanized antibody also comprises at least a portion of an
immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. In some
embodiments, a
humanized antibody contains both the light chain as well as at least the
variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4
regions of
the heavy chain. In some embodiments, a humanized antibody only contains a
humanized
light chain. In some embodiments, a humanized antibody only contains a
humanized heavy
chain. In specific embodiments, a humanized antibody only contains a humanized
variable
domain of a light chain and/or of a heavy chain.
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102031 The humanized antibody can be selected from any class of
immunoglobulins,
including IgM, IgG, IgD, IgA and IgE, and any isotype, including without
limitation IgG 1,
1gG2, 1gG3, and 1gG4. The humanized antibody may comprise sequences from more
than
one class or isotype, and particular constant domains may be selected to
optimize desired
effector functions using techniques well-known in the art.
10204] The framework and CDR regions of a humanized antibody need not
correspond
precisely to the parental sequences, e.g., the donor antibody CDR or the
consensus
framework may be mutagenized by substitution, insertion and/or deletion of at
least one
amino acid residue so that the CDR or framework residue at that site does not
correspond to
either the donor antibody or the consensus framework. In one embodiment, such
mutations,
however, will not be extensive. Usually, at least 90%, at least 95%, at least
98%, or at least
99% of the humanized antibody residues will correspond to those of the
parental FR and
CDR sequences. As used herein, the term "consensus framework" refers to the
framework
region in the consensus immunoglobulin sequence. As used herein, the term
"consensus
immunoglobulin sequence" refers to the sequence formed from the most
frequently occurring
amino acids (or nucleotides) in a family of related immunoglobulin sequences
(See e.g.,
Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)).
In a
family of immunoglobulins, each position in the consensus sequence is occupied
by the
amino acid occurring most frequently at that position in the family. If two
amino acids occur
equally frequently, either can be included in the consensus sequence.
102051 The humanized antibody may be designed to minimize unwanted
immunological
response toward rodent anti-human antibodies, which limits the duration and
effectiveness of
therapeutic applications of those moieties in human recipients. The humanized
antibody may
have one or more amino acid residues introduced into it from a source that is
non-human.
These non-human residues are often referred to as "import" residues, which are
typically
taken from a variable domain. Humanization may be performed by substituting
hypervariable region sequences for the corresponding sequences of a human
antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies wherein
substantially less
than an intact human variable domain has been substituted by the corresponding
sequence
from a non-human species. For example, see U.S. Patent No. 4,816,567, the
contents of
which are herein incorporated by reference. The humanized antibody may be a
human
antibody in which some hypervariable region residues, and possibly some FR
residues are
substituted by residues from analogous sites in rodent antibodies.
Humanization or
engineering of antibodies of the present invention can be performed using any
known
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method, such as but not limited to those described in U.S. Patent Nos.
5,723,323; 5,976,862;
5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352;
6,204,023;
6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.
[0206] The humanized antibody may retain high affinity for UCH-Li and other
favorable
biological properties. The humanized antibody may be prepared by a process of
analysis of
the parental sequences and various conceptual humanized products using three-
dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin
models are commonly available. Computer programs are available that illustrate
and display
probable three-dimensional conformational structures of selected candidate
immunoglobulin
sequences. Inspection of these displays permits analysis of the likely role of
the residues in
the functioning of the candidate immunoglobulin sequence, i.e., the analysis
of residues that
influence the ability of the candidate immunoglobulin to bind its antigen. In
this way, FR
residues can be selected and combined from the recipient and import sequences
so that the
desired antibody characteristics, such as increased affinity for UCH-L1, is
achieved. In
general, the hypervariable region residues may be directly and most
substantially involved in
influencing antigen binding.
102071 As an alternative to humanization, human antibodies (also referred to
herein as
"fully human antibodies") can be generated. For example, it is possible to
isolate human
antibodies from libraries via PROfusion and/or yeast related technologies. It
is also possible
to produce transgenic animals (e.g., mice that are capable, upon immunization,
of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin
production. For example, the homozygous deletion of the antibody heavy-chain
joining
region (JH) gene in chimeric and germ-line mutant mice results in complete
inhibition of
endogenous antibody production. Transfer of the human germ-line immunoglobulin
gene
array in such germ-line mutant mice will result in the production of human
antibodies upon
antigen challenge. The humanized or fully human antibodies may be prepared
according to
the methods described in U.S. Patent Nos. 5,770,429; 5,833,985; 5,837,243;
5,922,845;
6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;
6,682,928;
and 6,984,720, the contents each of which are herein incorporated by
reference.
e. Anti-UCH-L1 antibodies
[0208] Anti-UCH-L1 antibodies may be generated using the techniques described
above as
well as using routine techniques known in the art. In some embodiments, the
anti-UCH-L1
antibody may be an unconjugated UCH-Li antibody, such as UCH-Li antibodies
available
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from United State Biological (Catalog Number: 031320), Cell Signaling
Technology
(Catalog Number: 3524), Sigma-Aldrich (Catalog Number: HPA005993), Santa Cruz
Biotechnology, Inc. (Catalog Numbers: sc-58593 or sc-58594), R&D Systems
(Catalog
Number: MAB6007), Novus Biologicals (Catalog Number: NB600-1160), Biorbyt
(Catalog
Number: 0rb33715), Enzo Life Sciences, Inc. (Catalog Number: ADI-905-520-1),
Bio-Rad
(Catalog Number: VMA00004), BioVision (Catalog Number: 6130-50), Abcam
(Catalog
Numbers: ab75275 or ab104938), Invitrogen Antibodies (Catalog Numbers:
480012),
ThermoFisher Scientific (Catalog Numbers: MA1 -45079. MA5-17235. MA1-90008. or
MA 1-83428), EMD Millipore (Catalog Number: MABN48), or Sino Biological Inc.
(Catalog
Number: 50690-R011). The anti-UCH-L1 antibody may be conjugated to a
fluorophore,
such as conjugated UCH-L1 antibodies available from BioVision (Catalog Number:
6960-25)
or Aviva Systems Biology (Cat. Nos. OAAF01904-FITC).
6. Methods for Measuring the Level of GFAP
102091 In the methods described above, GFAP levels can be measured by any
means, such
as antibody dependent methods, such as immunoassays, protein
immunoprecipitation,
immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis,
or protein
immunostaining, electrophoresis analysis, a protein assay, a competitive
binding assay, a
functional protein assay, or chromatography or spectrometry methods, such as
high-
performance liquid chromatography (HPLC) or liquid chromatography¨mass
spectrometry
(LC/MS). Also, the assay can be employed in clinical chemistry format such as
would be
known by one skilled in the art.
102101 In some embodiments, measuring the level of GFAP includes contacting
the sample
with a first specific binding member and second specific binding member. In
some
embodiments the first specific binding member is a capture antibody and the
second specific
binding member is a detection antibody. In some embodiments, measuring the
level of GFAP
includes contacting the sample, either simultaneously or sequentially, in any
order: (1) a
capture antibody (e.g., GFAP-capture antibody), which binds to an epitope on
GFAP or
GFAP fragment to form a capture antibody-GFAP antigen complex (e.g., GFAP-
capture
antibody-GFAP antigen complex), and (2) a detection antibody (e.g., GFAP-
detection
antibody), which includes a detectable label and binds to an epitope on GFAP
that is not
bound by the capture antibody, to form a GFAP antigen-detection antibody
complex (e.g.,
GFAP antigen-GFAP-detection antibody complex), such that a capture antibody-
GFAP
antigen-detection antibody complex (e.g., GFAP-capture antibody-GFAP antigen-
GFAP-
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detection antibody complex) is formed, and measuring the amount or
concentration of GFAP
in the sample based on the signal generated by the detectable label in the
capture antibody-
GFAP antigen-detection antibody complex.
[0211] In some embodiments, the first specific binding member is immobilized
on a solid
support. In some embodiments, the second specific binding member is
immobilized on a
solid support. In some embodiments, the first specific binding member is a
GFAP antibody
as described below.
[0212] In some embodiments, the sample is diluted or undiluted. The sample can
be from
about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to
about 23
microliters, about 1 to about 22 microliters, about 1 to about 21 microliters,
about 1 to about
20 microliters, about 1 to about 18 microliters, about 1 to about 17
microliters, about 1 to
about 16 microliters, about 15 microliters or about 1 microliter, about 2
microliters, about 3
microliters, about 4 microliters, about 5 microliters, about 6 microliters,
about 7 microliters,
about 8 microliters, about 9 microliters, about 10 microliters, about 11
microliters, about 12
microliters, about 13 microliters, about 14 microliters, about 15 microliters,
about 16
microliters, about 17 microliters, about 18 microliters, about 19 microliters,
about 20
microliters, about 21 microliters, about 22 microliters, about 23 microliters,
about 24
microliters or about 25 microliters. In some embodiments, the sample is from
about 1 to
about 150 microliters or less or from about 1 to about 25 microliters or less.
[0213] Some instruments (such as, for example the Abbott Laboratories
instrument
ARCHITECT , and other core laboratory instruments) other than a point-of-care
device may
be capable of measuring levels of GFAP in a sample higher or greater than
25,000 pg/mL.
[0214] Other methods of detection include the use of or can be adapted for use
on a
nanopore device or nanowell device. Examples of nanopore devices are described
in
International Patent Publication No. WO 2016/161402, which is hereby
incorporated by
reference in its entirety. Examples of nanowell device are described in
International Patent
Publication No. WO 2016/161400, which is hereby incorporated by reference in
its entirety
7. GFAP Antibodies
[0215] The methods described herein may use an isolated antibody that
specifically binds to
Glial fibrillary acidic protein ("GFAP") (or fragments thereof), referred to
as "GFAP
antibody." The GFAP antibodies can be used to assess the GFAP status as a
measure of
traumatic brain injury, detect the presence of GFAP in a sample, quantify the
amount of
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GFAP present in a sample, or detect the presence of and quantify the amount of
GFAP in a
sample.
a. Glial fibrillary acidic protein (GFAP)
[0216] Glial fibrillary acidic protein (GFAP) is a 50 kDa intracytoplasmic
filamentous
protein that constitutes a portion of the cytoskeleton in astrocytes, and it
has proved to be the
most specific marker for cells of astrocytic origin. GFAP protein is encoded
by the GFAP
gene in humans. GFAP is the principal intermediate filament of mature
astrocytes. In the
central rod domain of the molecule, GFAP shares considerable structural
homology with the
other intermediate filaments. GFAP is involved in astrocyte motility and shape
by providing
structural stability to astrocytic processes. Glial fibrillary acidic protein
and its breakdown
products (GFAP-BDP) are brain-specific proteins released into the blood as
part of the
pathophysiological response after traumatic brain injury (TBI). Following
injury to the
human CNS caused by trauma, genetic disorders, or chemicals, astrocytes
proliferate and
show extensive hypertrophy of the cell body and processes, and GFAP is
markedly
upregulated. In contrast, with increasing astrocyte malignancy, there is a
progressive loss of
GFAP production. GFAP can also be detected in Schwann cells, enteric glia
cells, salivary
gland neoplasms, metastasizing renal carcinomas, epiglottic cartilage,
pituicytes, immature
oligodendrocytes, papillary meningiomas, and myoepithelial cells of the
breast.
[0217] Human GFAP may have the following amino acid sequence:
[0218] MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVD
FSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAK
EPTKLADVYQAELRELRLRLDQLTANS ARLEVERDNLAQDLATVRQKLQDETNLRL
EAENNLAAYRQEADEATLARLDLERKIESLEEElRFLRKIHEEEVRELQEQLARQQVH
VELDVAKPDLTAALKEIRTQYEAMAS SNMHEAEEWYRSKFADLTDAAARNAELLR
QAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLE
EEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRE
TSLDTKSVSEGHLKRN[VVKTVEMRDGEVIKESKQEHKDVM (SEQ ID NO: 2).
[0219] The human GFAP may be a fragment or variant of SEQ ID NO: 2. The
fragment of
GFAP may be between 5 and 400 amino acids, between 10 and 400 amino acids,
between 50
and 400 amino acids, between 60 and 400 amino acids, between 65 and 400 amino
acids,
between 100 and 400 amino acids, between 150 and 400 amino acids, between 100
and 300
amino acids, or between 200 and 300 amino acids in length. The fragment may
comprise a
contiguous number of amino acids from SEQ ID NO: 2. The human GFAP fragment or
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variant of SEQ ID NO: 2 may be a GFAP breakdown product (BDP). The GFAP BDP
may
be 38 kDa, 42 kDa (fainter 41 kDa), 47 kDa (fainter 45 kDa); 25 kDa (fainter
23 kDa); 19
kDa, or 20 kDa.
b. GFAP-Recognizing Antibody
[0220] The antibody is an antibody that binds to GFAP, a fragment thereof, an
epitope of
GFAP, or a variant thereof. The antibody may be a fragment of the anti-GFAP
antibody or a
variant or a derivative thereof. The antibody may be a polyclonal or
monoclonal antibody.
The antibody may be a chimeric antibody, a single chain antibody, an affinity
matured
antibody, a human antibody, a humanized antibody, a fully human antibody or an
antibody
fragment, such as a Fab fragment, or a mixture thereof. Antibody fragments or
derivatives
may comprise F(ab')2, Fv or scFv fragments. The antibody derivatives can be
produced by
peptidomimetics. Further, techniques described for the production of single
chain antibodies
can be adapted to produce single chain antibodies.
[0221] The anti-GFAP antibodies may be a chimeric anti-GFAP or humanized anti-
GFAP
antibody. In one embodiment, both the humanized antibody and chimeric antibody
are
monovalent. In one embodiment, both the humanized antibody and chimeric
antibody
comprise a single Fab region linked to an Fc region.
[0222] Human antibodies may be derived from phage-display technology or from
transgenic mice that express human immunoglobulin genes. The human antibody
may be
generated as a result of a human in vivo immune response and isolated. See,
for example,
Funaro et al., BMC Biotechnology, 2008(8):85. Therefore, the antibody may be a
product of
the human and not animal repertoire. Because it is of human origin, the risks
of reactivity
against self-antigens may be minimized. Alternatively, standard yeast display
libraries and
display technologies may be used to select and isolate human anti-GFAP
antibodies. For
example, libraries of naïve human single chain variable fragments (scFv) may
be used to
select human anti-GFAP antibodies. Transgenic animals may be used to express
human
antibodies.
[0223] Humanized antibodies may be antibody molecules from non-human species
antibody that binds the desired antigen having one or more complementarity
determining
regions (CDRs) from the non-human species and framework regions from a human
immunoglobulin molecule.
[0224] The antibody is distinguishable from known antibodies in that it
possesses different
biological function(s) than those known in the art.
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(1) Epitope
[0225] The antibody may immunospecifically bind to GFAP (SEQ ID NO: 2), a
fragment
thereof, or a variant thereof. The antibody may immunospecifically recognize
and bind at
least three amino acids, at least four amino acids, at least five amino acids,
at least six amino
acids, at least seven amino acids, at least eight amino acids, at least nine
amino acids, or at
least ten amino acids within an epitope region. The antibody may
immunospecifically
recognize and bind to an epitope that has at least three contiguous amino
acids, at least four
contiguous amino acids, at least five contiguous amino acids, at least six
contiguous amino
acids, at least seven contiguous amino acids, at least eight contiguous amino
acids, at least
nine contiguous amino acids, or at least ten contiguous amino acids of an
epitope region.
c. Antibody Preparation/Production
[0226] Antibodies may be prepared by any of a variety of techniques, including
those well
known to those skilled in the art. In general, antibodies can be produced by
cell culture
techniques, including the generation of monoclonal antibodies via conventional
techniques,
or via transfection of antibody genes, heavy chains, and/or light chains into
suitable bacterial
or mammalian cell hosts, in order to allow for the production of antibodies,
wherein the
antibodies may be recombinant. The various forms of the term "transfection"
are intended to
encompass a wide variety of techniques commonly used for the introduction of
exogenous
DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-
phosphate
precipitation, DEAE-dextran transfection and the like. Although it is possible
to express the
antibodies in either prokaryotic or eukaryotic host cells, expression of
antibodies in
eukaryotic cells is preferable, and most preferable in mammalian host cells,
because such
eukaryotic cells (and in particular mammalian cells) are more likely than
prokaryotic cells to
assemble and secrete a properly folded and immunologically active antibody.
[0227] Exemplary mammalian host cells for expressing the recombinant
antibodies include
Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in
Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), used with a DHFR
selectable
marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621
(1982), NSO
myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors
encoding
antibody genes are introduced into mammalian host cells, the antibodies are
produced by
culturing the host cells for a period of time sufficient to allow for
expression of the antibody
in the host cells or, more preferably, secretion of the antibody into the
culture medium in
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which the host cells are grown. Antibodies can be recovered from the culture
medium using
standard protein purification methods.
10228] Host cells can also be used to produce functional antibody fragments,
such as Fab
fragments or scFv molecules. It will be understood that variations on the
above procedure
may be performed. For example, it may be desirable to transfect a host cell
with DNA
encoding functional fragments of either the light chain and/or the heavy chain
of an antibody.
Recombinant DNA technology may also be used to remove some, or all, of the DNA
encoding either or both of the light and heavy chains that is not necessary
for binding to the
antigens of interest. The molecules expressed from such truncated DNA
molecules are also
encompassed by the antibodies. In addition, bifunctional antibodies may be
produced in
which one heavy and one light chain are an antibody (i.e., binds human GFAP)
and the other
heavy and light chain are specific for an antigen other than human GFAP by
crosslinking an
antibody to a second antibody by standard chemical crosslinking methods.
102291 In a preferred system for recombinant expression of an antibody, or
antigen-binding
portion thereof, a recombinant expression vector encoding both the antibody
heavy chain and
the antibody light chain is introduced into dhfr-CHO cells by calcium
phosphate-mediated
transfe,ction. Within the recombinant expression vector, the antibody heavy
and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory
elements to
drive high levels of transcription of the genes. The recombinant expression
vector also
carries a DHPR gene, which allows for selection of CHO cells that have been
transfected
with the vector using methotrexate selection/amplification. The selected
transformant host
cells are cultured to allow for expression of the antibody heavy and light
chains and intact
antibody is recovered from the culture medium. Standard molecular biology
techniques are
used to prepare the recombinant expression vector, transfect the host cells,
select for
transformants, culture the host cells, and recover the antibody from the
culture medium. Still
further, the method of synthesizing a recombinant antibody may be by culturing
a host cell in
a suitable culture medium until a recombinant antibody is synthesized. The
method can
further comprise isolating the recombinant antibody from the culture medium.
102301 Methods of preparing monoclonal antibodies involve the preparation of
immortal
cell lines capable of producing antibodies having the desired specificity.
Such cell lines may
be produced from spleen cells obtained from an immunized animal. The animal
may be
immunized with GFAP or a fragment and/or variant thereof. The peptide used to
immunize
the animal may comprise amino acids encoding human Fc, for example the
fragment
crystallizable region or tail region of human antibody. The spleen cells may
then be
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immortalized by, for example, fusion with a myeloma cell fusion partner. A
variety of fusion
techniques may be employed. For example, the spleen cells and myeloma cells
may be
combined with a nonionic detergent for a few minutes and then plated at low
density on a
selective medium that supports that growth of hybrid cells, but not myeloma
cells. One such
technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another
technique
includes electrofusion. After a sufficient time, usually about 1 to 2 weeks,
colonies of
hybrids are observed. Single colonies are selected and their culture
supernatants tested for
binding activity against the polypeptide. Hybridomas having high reactivity
and specificity
may be used.
102311 Monoclonal antibodies may be isolated from the supernatants of growing
hybridoma
colonies. In addition, various techniques may be employed to enhance the
yield, such as
injection of the hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host,
such as a mouse. Monoclonal antibodies may then be harvested from the ascites
fluid or the
blood. Contaminants may be removed from the antibodies by conventional
techniques, such
as chromatography, gel filtration, precipitation, and extraction. Affinity
chromatography is
an example of a method that can be used in a process to purify the antibodies.
102321 The proteolytic enzyme papain preferentially cleaves IgG molecules to
yield several
fragments, two of which (the F(ab) fragments) each comprise a covalent
heterodimer that
includes an intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to
provide several fragments, including the F(ab')2 fragment, which comprises
both antigen-
binding sites.
102331 The Fv fragment can be produced by preferential proteolytic cleavage of
an IgM,
and on rare occasions IgG or IgA immunoglobulin molecules. The Fv fragment may
be
derived using recombinant techniques. The Fv fragment includes a non-covalent
VH:VL
heterodimer including an antigen-binding site that retains much of the antigen
recognition
and binding capabilities of the native antibody molecule.
[0234] The antibody, antibody fragment, or derivative may comprise a heavy
chain and a
light chain complementarity determining region ("CDR") set, respectively
interposed
between a heavy chain and a light chain framework ("FR") set which provide
support to the
CDRs and define the spatial relationship of the CDRs relative to each other.
The CDR set
may contain three hypervariable regions of a heavy or light chain V region.
[0235] Other suitable methods of producing or isolating antibodies of the
requisite
specificity can be used, including, but not limited to, methods that select
recombinant
antibody from a peptide or protein library (e.g., but not limited to, a
bacteriophage, ribosome,
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oligonucleotide, RNA, cDNA, yeast or the like, display library); e.g., as
available from
various commercial vendors such as Cambridge Antibody Technologies
(Cambridgeshire,
UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK)
BioInvent (Lund, Sweden), using methods known in the art. See U.S. Patent Nos.
4,704,692;
5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative
methods rely
upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1997)
Microbiol.
Immunol. 41:901-907; Sandhu et al. (1996) Grit. Rev. Biotechnol. 16:95-118;
Eren et al.
(1998) Immunol. 93:154-161) that are capable of producing a repertoire of
human antibodies,
as known in the art and/or as described herein. Such techniques, include, but
are not limited
to, ribosome display (Hanes et al. (1997) Proc. Nail. Acad. Sci. USA, 94:4937-
4942; Hanes et
al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody
producing
technologies (e.g., selected lymphocyte antibody method ("SLAM") (U.S. Patent
No.
5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996)
Proc. Natl.
Acad. Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell et
al. (1990)
Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al.
(1995) J. Imm.
Meth. 182:155-163; Kenny et al. (1995) Bio/Technol. 13:787-790); B-cell
selection
(Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).
[0236] An affinity matured antibody may be produced by any one of a number of
procedures that are known in the art. For example, see Marks et al.,
BioTechnology,10: 779-
783 (1992) describes affinity maturation by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by Barbas et al.,
Proc. Nat.
Acad. Sci. USA, 91: 3809-3813 (1994); Schier et al., Gene, 169: 147-155
(1995); Yelton et
al., J. Immunol 155: 1994-2004 (1995); Jackson et al., J. Immunol., 154(7):
3310-3319
(1995); Hawkins et al, J. Mol. Biol., 226: 889-896 (1992). Selective mutation
at selective
mutagenesis positions and at contact or hypermutation positions with an
activity enhancing
amino acid residue is described in U.S. Patent No. 6,914,128 Bl.
[0237] Antibody variants can also be prepared using delivering a
polynucleotide encoding
an antibody to a suitable host such as to provide transgenic animals or
mammals, such as
goats, cows, horses, sheep, and the like, that produce such antibodies in
their milk. These
methods are known in the art and are described for example in U.S. Patent Nos.
5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.
[0238] Antibody variants also can be prepared by delivering a polynucleotide
to provide
transgenic plants and cultured plant cells (e.g., but not limited to tobacco,
maize, and
duckweed) that produce such antibodies, specified portions or variants in the
plant parts or in
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cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.
Microbiol. Immunol.
240:95-118 and references cited therein, describe the production of transgenic
tobacco leaves
expressing large amounts of recombinant proteins, e.g., using an inducible
promoter.
Transgenic maize have been used to express mammalian proteins at commercial
production
levels, with biological activities equivalent to those produced in other
recombinant systems or
purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol.
(1999) 464:127-
147 and references cited therein. Antibody variants have also been produced in
large
amounts from transgenic plant seeds including antibody fragments, such as
single chain
antibodies (scFvs), including tobacco seeds and potato tubers. See, e.g.,
Conrad et al. (1998)
Plant Mol. Biol. 38:101-109 and reference cited therein. Thus, antibodies can
also be
produced using transgenic plants, according to known methods.
[0239] Antibody derivatives can be produced, for example, by adding exogenous
sequences
to modify immunogenicity or reduce, enhance or modify binding, affinity, on-
rate, off-rate,
avidity, specificity, half-life, or any other suitable characteristic.
Generally, part or all of the
non-human or human CDR sequences are maintained while the non-human sequences
of the
variable and constant regions are replaced with human or other amino acids.
102401 Small antibody fragments may be diabodies having two antigen-binding
sites,
wherein fragments comprise a heavy chain variable domain (VH) connected to a
light chain
variable domain (VL) in the same polypeptide chain (VH VL). See for example,
EP 404,097;
WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad. ,S'ci. USA 90:6444-
6448. By
using a linker that is too short to allow pairing between the two domains on
the same chain,
the domains are forced to pair with the complementary domains of another chain
and create
two antigen-binding sites. See also, U.S. Patent No. 6,632,926 to Chen et al.
which is hereby
incorporated by reference in its entirety and discloses antibody variants that
have one or more
amino acids inserted into a hypervariable region of the parent antibody and a
binding affinity
for a target antigen which is at least about two fold stronger than the
binding affinity of the
parent antibody for the antigen.
[0241] The antibody may be a linear antibody. The procedure for making a
linear antibody
is known in the art and described in Zapata et al. (1995) Protein Eng.
8(10):1057-1062.
Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-
CH1) which
form a pair of antigen binding regions. Linear antibodies can be bispecific or
monospecific.
[0242] The antibodies may be recovered and purified from recombinant cell
cultures by
known methods including, but not limited to, protein A purification, ammonium
sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
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phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxyl apati te chromatography and lectin chromatography.
High
performance liquid chromatography ("HPLC") can also be used for purification.
[0243] It may be useful to detectably label the antibody. Methods for
conjugating
antibodies to these agents are known in the art. For the purpose of
illustration only,
antibodies can be labeled with a detectable moiety such as a radioactive atom,
a
chromophore, a fluorophore, or the like. Such labeled antibodies can be used
for diagnostic
techniques, either in vivo, or in an isolated test sample. They can be linked
to a cytokine, to a
ligand, to another antibody. Suitable agents for coupling to antibodies to
achieve an anti-
tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor
Necrosis Factor
(TNF); photosensitizers, for use in photodynamic therapy, including aluminum
(III)
phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine;
radionuclides, such as
iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi),
technetium-
99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such
as
doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,
neocarzinostatin, and
carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin,
pseudomonas
exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A
(deglycosylated ricin A and
native ricin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja atm), and
gelonin (a
plant toxin); ribosome inactivating proteins from plants, bacteria and fungi,
such as
restrictocin (a ribosome inactivating protein produced by Aspergillus
restrictus), saporin (a
ribosome inactivating protein from Saponaria officinalis), and RNase; tyrosine
kinase
inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing
anti cystic
agents (e.g., antisense oligonucleotides, plasmids which encode for toxins,
methotrexate,
etc.); and other antibodies or antibody fragments, such as F(ab).
[0244] Antibody production via the use of hybridoma technology, the selected
lymphocyte
antibody method (SLAM), transgenic animals, and recombinant antibody libraries
is
described in more detail below.
(1) Anti-GFAP Monoclonal Antibodies Using Hybridoma Technology
[0245] Monoclonal antibodies can be prepared using a wide variety of
techniques known in
the art including the use of hybridoma, recombinant, and phage display
technologies, or a
combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, second edition, (Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, 1988); Hammerling, et al., In Monoclonal Antibodies and T-
Cell
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Hybridomus, (Elsevier, N.Y., 1981). It is also noted that the term "monoclonal
antibody" as
used herein is not limited to antibodies produced through hybridoma
technology. The term
"monoclonal antibody" refers to an antibody that is derived from a single
clone, including
any eukaryotic, prokaryotic, or phage clone, and not the method by which it is
produced.
102461 Methods of generating monoclonal antibodies as well as antibodies
produced by the
method may comprise culturing a hybridoma cell secreting an antibody of the
invention
wherein, preferably, the hybridoma is generated by fusing splenocytes isolated
from an
animal, e.g., a rat or a mouse, immunized with GFAP with myeloma cells and
then screening
the hybridomas resulting from the fusion for hybridoma clones that secrete an
antibody able
to bind a polypeptide of the invention. Briefly, rats can be immunized with a
GFAP antigen.
In a preferred embodiment, the GFAP antigen is administered with an adjuvant
to stimulate
the immune response. Such adjuvants include complete or incomplete Freund's
adjuvant,
RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such
adjuvants may
protect the polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may
contain substances that stimulate the host to secrete factors that are
chemotactic for
macrophages and other components of the immune system. Preferably, if a
polypeptide is
being administered, the immunization schedule will involve two or more
administrations of
the polypeptide, spread out over several weeks; however, a single
administration of the
polypeptide may also be used.
102471 After immunization of an animal with a GFAP antigen, antibodies and/or
antibody-
producing cells may be obtained from the animal. An anti-GFAP antibody-
containing serum
is obtained from the animal by bleeding or sacrificing the animal. The serum
may be used as
it is obtained from the animal, an immunoglobulin fraction may be obtained
from the serum,
or the anti-GFAP antibodies may be purified from the serum. Serum or
immunoglobulins
obtained in this manner are polyclonal, thus having a heterogeneous array of
properties.
102481 Once an immune response is detected, e.g., antibodies specific for the
antigen GFAP
are detected in the rat serum, the rat spleen is harvested and splenocytes
isolated. The
splenocytes are then fused by well-known techniques to any suitable myeloma
cells, for
example, cells from cell line SP20 available from the American Type Culture
Collection
(ATCC, Manassas, Va., US). Hybridomas are selected and cloned by limited
dilution. The
hybridoma clones are then assayed by methods known in the art for cells that
secrete
antibodies capable of binding GFAP. Ascites fluid, which generally contains
high levels of
antibodies, can be generated by immunizing rats with positive hybridoma
clones.
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[0249] In another embodiment, antibody-producing immortalized hybridomas may
be
prepared from the immunized animal. After immunization, the animal is
sacrificed, and the
splenic B cells are fused to immortalized myeloma cells as is well known in
the art. See, e.g.,
Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not
secrete
immunoglobulin polypeptides (a non-secretory cell line). After fusion and
antibiotic
selection, the hybridomas are screened using GFAP, or a portion thereof, or a
cell expressing
GFAP. In a preferred embodiment, the initial screening is performed using an
enzyme-linked
immunosorbent assay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA.
An
example of ELISA screening is provided in PCT Publication No. WO 00/37504.
[0250] Anti-GFAP antibody-producing hybridomas are selected, cloned, and
further
screened for desirable characteristics, including robust hybridoma growth,
high antibody
production, and desirable antibody characteristics. Hybridomas may be cultured
and
expanded in vivo in syngeneic animals, in animals that lack an immune system,
e.g., nude
mice, or in cell culture in vitro. Methods of selecting, cloning and expanding
hybridomas are
well known to those of ordinary skill in the art.
[0251] In a preferred embodiment, hybridomas are rat hybridomas. In another
embodiment,
hybridomas are produced in a non-human, non-rat species such as mice, sheep,
pigs, goats,
cattle, or horses. In yet another preferred embodiment, the hybridomas are
human
hybridomas, in which a human non-secretory myeloma is fused with a human cell
expressing
an anti-GFAP antibody.
[0252] Antibody fragments that recognize specific epitopes may be generated by
known
techniques. For example, Fab and F(ab')2 fragments of the invention may be
produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain
(to
produce two identical Fab fragments) or pepsin (to produce an F(ab')2
fragment). A F(ab')2
fragment of an IgG molecule retains the two antigen-binding sites of the
larger ("parent") IgG
molecule, including both light chains (containing the variable light chain and
constant light
chain regions), the CH1 domains of the heavy chains, and a disulfide-forming
hinge region of
the parent IgG molecule. Accordingly, an F(ab')2 fragment is still capable of
crosslinking
antigen molecules like the parent IgG molecule.
(2) Anti-GFAP Monoclonal Antibodies Using SLAM
[0253] In another aspect of the invention, recombinant antibodies are
generated from single,
isolated lymphocytes using a procedure referred to in the art as the selected
lymphocyte
antibody method (SLAM), as described in U.S. Patent No. 5,627,052; PCT
Publication No.
WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93: 7843-7848
(1996). In this
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method, single cells secreting antibodies of interest, e.g., lymphocytes
derived from any one
of the immunized animals are screened using an antigen-specific hemolytic
plaque assay,
wherein the antigen GFAP, a subunit of GFAP, or a fragment thereof, is coupled
to sheep red
blood cells using a linker, such as biotin, and used to identify single cells
that secrete
antibodies with specificity for GFAP. Following identification of antibody-
secreting cells of
interest, heavy- and light-chain variable region cDNAs are rescued from the
cells by reverse
transcriptase-PCR (RT-PCR) and these variable regions can then be expressed,
in the context
of appropriate immunoglobulin constant regions (e.g., human constant regions),
in
mammalian host cells, such as COS or CHO cells. The host cells transfected
with the
amplified immunoglobulin sequences, derived from in vivo selected lymphocytes,
can then
undergo further analysis and selection in vitro, for example, by panning the
transfected cells
to isolate cells expressing antibodies to GFAP. The amplified immunoglobulin
sequences
further can be manipulated in vitro, such as by in vitro affinity maturation
method. See, for
example, PCT Publication No. WO 97/29131 and PCT Publication No. WO 00/56772.
(3) Anti-GFAP Monoclonal Antibodies Using Transgenic Animals
[0254] In another embodiment of the invention, antibodies are produced by
immunizing a
non-human animal comprising some, or all, of the human immunoglobulin locus
with a
GFAP antigen. In an embodiment, the non-human animal is a XENOMOUSE
transgenic
mouse, an engineered mouse strain that comprises large fragments of the human
immunoglobulin loci and is deficient in mouse antibody production. See, e.g.,
Green et al.,
Nature Genetics, 7: 13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598;
5,985,615;
5,998,209; 6,075,181; 6,091,001; 6,114,598; and 6,130,364. See also PCT
Publication Nos.
WO 91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO 98/24893;
WO 98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The
XENOMOUSE transgenic mouse produces an adult-like human repertoire of fully
human
antibodies, and generates antigen-specific human monoclonal antibodies. The
XENOMOUSE transgenic mouse contains approximately 80% of the human antibody
repertoire through introduction of megabase sized, germline configuration YAC
fragments of
the human heavy chain loci and x light chain loci. See Mendez et al., Nature
Genetics, 15:
146-156 (1997), Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the
disclosures of
which are hereby incorporated by reference.
(4) Anti-GFAP Monoclonal Antibodies Using Recombinant Antibody Libraries
[0255] In vitro methods also can be used to make the antibodies of the
invention, wherein
an antibody library is screened to identify an antibody having the desired
GFAP -binding
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specificity. Methods for such screening of recombinant antibody libraries are
well known in
the art and include methods described in, for example, U.S. Patent No.
5,223,409 (Ladner et
al.); PCT Publication No. WO 92/18619 (Kang et al.); PCT Publication No. WO
91/17271
(Dower et al.); PCT Publication No. WO 92/20791 (Winter et al.); PCT
Publication No. WO
92/15679 (Markland et al.); PCT Publication No. WO 93/01288 (Breitling et
al.); PCT
Publication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO
92/09690
(Garrard et al.); Fuchs et al., Bio/Technology, 9: 1369-1372 (1991); Hay et
al., Hum. Antibod.
Hybridomas, 3: 81-85 (1992); Huse et al.. Science, 246: 1275-1281 (1989);
McCafferty et al.,
Nature, 348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993);
Hawkins et al.,
J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628
(1991); Gram et al.,
Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992); Garrard et al.,
Bio/Technology, 9: 1373-
1377 (1991); Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas
et al.,
Proc. Natl. Acad. Sc!. USA, 88: 7978-7982 (1991); U.S. Patent Application
Publication No.
2003/0186374; and PCT Publication No. WO 97/29131, the contents of each of
which are
incorporated herein by reference.
102561 The recombinant antibody library may be from a subject immunized with
GFAP, or
a portion of GFAP. Alternatively, the recombinant antibody library may be from
a naive
subject, i.e., one who has not been immunized with GFAP, such as a human
antibody library
from a human subject who has not been immunized with human GFAP. Antibodies of
the
invention are selected by screening the recombinant antibody library with the
peptide
comprising human GFAP to thereby select those antibodies that recognize GFAP.
Methods
for conducting such screening and selection are well known in the art, such as
described in
the references in the preceding paragraph. To select antibodies of the
invention having
particular binding affinities for GFAP, such as those that dissociate from
human GFAP with a
particular Koff rate constant, the art-known method of surface plasmon
resonance can be used
to select antibodies having the desired Koff rate constant. To select
antibodies of the invention
having a particular neutralizing activity for GFAP, such as those with a
particular IC50,
standard methods known in the art for assessing the inhibition of GFAP
activity may be used.
102571 In one aspect, the invention pertains to an isolated antibody, or an
antigen-binding
portion thereof, that binds human GFAP. Preferably, the antibody is a
neutralizing antibody.
In various embodiments, the antibody is a recombinant antibody or a monoclonal
antibody.
[0258] For example, antibodies can also be generated using various phage
display methods
known in the art. In phage display methods, functional antibody domains are
displayed on the
surface of phage particles which carry the polynucleotide sequences encoding
them. Such
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phage can be utilized to display antigen-binding domains expressed from a
repertoire or
combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding
domain that binds the antigen of interest can be selected or identified with
antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or bead. Phage
used in these
methods are typically filamentous phage including fd and M13 binding domains
expressed
from phage with Fab, Fv, or disulfide stabilized Fv antibody domains
recombinantly fused to
either the phage gene III or gene VIII protein. Examples of phage display
methods that can be
used to make the antibodies include those disclosed in Brinkmann et al., J.
Immunol.
Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186
(1995);
Kettleborough et al., Fur. J. Immunol., 24: 952-958 (1994); Persic et al.,
Gene, 187: 9-18
(1997); Burton et al., Advances in Immunology, 57: 191-280 (1994); PCT
Publication No.
WO 92/01047; PCT Publication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos.
5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.
[0259] As described in the above references, after phage selection, the
antibody coding
regions from the phage can be isolated and used to generate whole antibodies
including
human antibodies or any other desired antigen binding fragment, and expressed
in any
desired host, including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as
described in detail below. For example, techniques to recombinantly produce
Fab, Fab', and
F(ab')7 fragments can also be employed using methods known in the art such as
those
disclosed in PCT publication No. WO 92/22324; Mullinax et al., BioTechniques,
12(6): 864-
869 (1992); Sawai et al., Am. I Reprod. Immunol., 34: 26-34 (1995); and Better
et al.,
Science, 240: 1041-1043 (1988). Examples of techniques which can be used to
produce
single-chain Fvs and antibodies include those described in U.S. Patent Nos.
4,946,778 and
5,258,498; Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et
al., Proc. Natl.
Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science, 240: 1038-
1041 (1988).
[0260] Alternative to screening of recombinant antibody libraries by phage
display, other
methodologies known in the art for screening large combinatorial libraries can
be applied to
the identification of antibodies of the invention. One type of alternative
expression system is
one in which the recombinant antibody library is expressed as RNA-protein
fusions, as
described in PCT Publication No. WO 98/31700 (Szostak and Roberts), and in
Roberts and
Szostak, Proc. Natl. Acad. Sci. USA, 94: 12297-12302 (1997). In this system, a
covalent
fusion is created between an mRNA and the peptide or protein that it encodes
by in vitro
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translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor
antibiotic, at their
3' end. Thus, a specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a
combinatorial library) based on the properties of the encoded peptide or
protein, e.g.,
antibody, or portion thereof, such as binding of the antibody, or portion
thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or portions
thereof,
recovered from screening of such libraries can be expressed by recombinant
means as
described above (e.g., in mammalian host cells) and, moreover, can be
subjected to further
affinity maturation by either additional rounds of screening of mRNA-peptide
fusions in
which mutations have been introduced into the originally selected sequence(s),
or by other
methods for affinity maturation in vitro of recombinant antibodies, as
described above. A
preferred example of this methodology is PROfusion display technology.
[0261] In another approach, the antibodies can also be generated using yeast
display
methods known in the art. In yeast display methods, genetic methods are used
to tether
antibody domains to the yeast cell wall and display them on the surface of
yeast. Such yeast
can be utilized to display antigen-binding domains expressed from a repertoire
or
combinatorial antibody library (e.g., human or marine). Examples of yeast
display methods
that can be used to make the antibodies include those disclosed in U.S. Patent
No. 6,699,658
(Wittrup et al.) incorporated herein by reference.
d. Production of Recombinant GFAP Antibodies
[0262] Antibodies may be produced by any of a number of techniques known in
the art.
For example, expression from host cells, wherein expression vector(s) encoding
the heavy
and light chains is (are) transfected into a host cell by standard techniques.
The various forms
of the term "transfection" are intended to encompass a wide variety of
techniques commonly
used for the introduction of exogenous DNA into a prokaryotic or eukaryotic
host cell, e.g.,
electroporation, calcium-phosphate precipitation, DEAE-dextran transfection,
and the like.
Although it is possible to express the antibodies of the invention in either
prokaryotic or
eukaryotic host cells, expression of antibodies in eukaryotic cells is
preferable, and most
preferable in mammalian host cells, because such eukaryotic cells (and in
particular
mammalian cells) are more likely than prokaryotic cells to assemble and
secrete a properly
folded and immunologically active antibody.
[0263] Exemplary mammalian host cells for expressing the recombinant
antibodies of the
invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells,
described
in Urlaub and ChasM, Proc. Nail. Acad. Sci. USA, 77: 4216-4220 (1980), used
with a DHFR
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selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol.,
159: 601-621
(1982), NSO myeloma cells, COS cells, and SP2 cells. When recombinant
expression vectors
encoding antibody genes are introduced into mammalian host cells, the
antibodies are
produced by culturing the host cells for a period of time sufficient to allow
for expression of
the antibody in the host cells or, more preferably, secretion of the antibody
into the culture
medium in which the host cells are grown. Antibodies can be recovered from the
culture
medium using standard protein purification methods.
102641 Host cells can also be used to produce functional antibody fragments,
such as Fab
fragments or scFv molecules. It will be understood that variations on the
above procedure
may be performed. For example, it may be desirable to transfect a host cell
with DNA
encoding functional fragments of either the light chain and/or the heavy chain
of an antibody
of this invention. Recombinant DNA technology may also be used to remove some,
or all, of
the DNA encoding either or both of the light and heavy chains that is not
necessary for
binding to the antigens of interest. The molecules expressed from such
truncated DNA
molecules are also encompassed by the antibodies of the invention. In
addition, bifunctional
antibodies may be produced in which one heavy and one light chain are an
antibody of the
invention (i.e., binds human GFAP) and the other heavy and light chain are
specific for an
antigen other than human GFAP by crosslinking an antibody of the invention to
a second
antibody by standard chemical crosslinking methods.
10265] In a preferred system for recombinant expression of an antibody, or
antigen-binding
portion thereof, of the invention, a recombinant expression vector encoding
both the antibody
heavy chain and the antibody light chain is introduced into dhfr-CHO cells by
calcium
phosphate-mediated transfection. Within the recombinant expression vector, the
antibody
heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP
promoter
regulatory elements to drive high levels of transcription of the genes. The
recombinant
expression vector also carries a DHFR gene, which allows for selection of CHO
cells that
have been transfected with the vector using methotrexate
selection/amplification. The
selected transformant host cells are cultured to allow for expression of the
antibody heavy
and light chains and intact antibody is recovered from the culture medium.
Standard
molecular biology techniques are used to prepare the recombinant expression
vector, transfect
the host cells, select for transformants, culture the host cells, and recover
the antibody from
the culture medium. Still further, the invention provides a method of
synthesizing a
recombinant antibody of the invention by culturing a host cell of the
invention in a suitable
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culture medium until a recombinant antibody of the invention is synthesized.
The method
can further comprise isolating the recombinant antibody from the culture
medium.
(1) Humanized Antibody
102661 The humanized antibody may be an antibody or a variant, derivative,
analog or
portion thereof which immunospecifically binds to an antigen of interest and
which
comprises a framework (FR) region having substantially the amino acid sequence
of a human
antibody and a complementary determining region (CDR) having substantially the
amino
acid sequence of a non-human antibody. The humanized antibody may be from a
non-human
species antibody that binds the desired antigen having one or more
complementarity
determining regions (CDRs) from the non-human species and framework regions
from a
human immunoglobulin molecule.
102671 As used herein, the term "substantially" in the context of a CDR refers
to a CDR
having an amino acid sequence at least 90%, at least 95%, at least 98% or at
least 99%
identical to the amino acid sequence of a non-human antibody CDR. A humanized
antibody
comprises substantially all of at least one, and typically two, variable
domains (Fab, Fab',
F(ab'),, FabC, Fv) in which all or substantially all of the CDR regions
correspond to those of
a non-human immunoglobulin (i.e., donor antibody) and all or substantially all
of the
framework regions are those of a human immunoglobulin consensus sequence.
According to
one aspect, a humanized antibody also comprises at least a portion of an
immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. In some
embodiments, a
humanized antibody contains both the light chain as well as at least the
variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4
regions of
the heavy chain. In some embodiments, a humanized antibody only contains a
humanized
light chain. In some embodiments, a humanized antibody only contains a
humanized heavy
chain. In specific embodiments, a humanized antibody only contains a humanized
variable
domain of a light chain and/or of a heavy chain.
102681 The humanized antibody can be selected from any class of
immunoglobulins,
including IgM, IgG, IgD, IgA and IgE, and any isotype, including without
limitation IgG 1,
IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more
than
one class or isotype, and particular constant domains may be selected to
optimize desired
effector functions using techniques well-known in the art.
102691 The framework and CDR regions of a humanized antibody need not
correspond
precisely to the parental sequences, e.g., the donor antibody CDR or the
consensus
framework may be mutagenized by substitution, insertion and/or deletion of at
least one
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amino acid residue so that the CDR or framework residue at that site does not
correspond to
either the donor antibody or the consensus framework. In one embodiment, such
mutations,
however, will not be extensive. Usually, at least 90%, at least 95%, at least
98%, or at least
99% of the humanized antibody residues will correspond to those of the
parental FR and
CDR sequences. As used herein, the term "consensus framework" refers to the
framework
region in the consensus immunoglobulin sequence. As used herein, the term
"consensus
immunoglobulin sequence" refers to the sequence formed from the most
frequently occurring
amino acids (or nucleotides) in a family of related immunoglobulin sequences
(See e.g.,
Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)).
In a
family of immunoglobulins, each position in the consensus sequence is occupied
by the
amino acid occurring most frequently at that position in the family. If two
amino acids occur
equally frequently, either can be included in the consensus sequence.
102701 The humanized antibody may be designed to minimize unwanted
immunological
response toward rodent anti-human antibodies, which limits the duration and
effectiveness of
therapeutic applications of those moieties in human recipients. The humanized
antibody may
have one or more amino acid residues introduced into it from a source that is
non-human.
These non-human residues are often referred to as "import" residues, which are
typically
taken from a variable domain. Humanization may be performed by substituting
hypervari able region sequences for the corresponding sequences of a human
antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies wherein
substantially less
than an intact human variable domain has been substituted by the corresponding
sequence
from a non-human species. For example, see U.S. Patent No. 4,816,567, the
contents of
which are herein incorporated by reference. The humanized antibody may be a
human
antibody in which some hypervariable region residues, and possibly some FR
residues are
substituted by residues from analogous sites in rodent antibodies.
Humanization or
engineering of antibodies of the present invention can be performed using any
known
method, such as but not limited to those described in U.S. Patent Nos.
5,723,323; 5,976,862;
5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352;
6,204,023;
6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.
[0271] The humanized antibody may retain high affinity for GFAP and other
favorable
biological properties. The humanized antibody may be prepared by a process of
analysis of
the parental sequences and various conceptual humanized products using three-
dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin
models are commonly available. Computer programs are available that illustrate
and display
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probable three-dimensional conformational structures of selected candidate
immunoglobulin
sequences. Inspection of these displays permits analysis of the likely role of
the residues in
the functioning of the candidate immunoglobulin sequence, i.e., the analysis
of residues that
influence the ability of the candidate immunoglobulin to bind its antigen. In
this way, FR
residues can be selected and combined from the recipient and import sequences
so that the
desired antibody characteristics, such as increased affinity for GFAP, is
achieved. In general,
the hypervariable region residues may be directly and most substantially
involved in
influencing antigen binding.
102721 As an alternative to humanization, human antibodies (also referred to
herein as
"fully human antibodies") can be generated. For example, it is possible to
isolate human
antibodies from libraries via PROfusion and/or yeast related technologies. It
is also possible
to produce transgenic animals (e.g. mice that are capable, upon immunization,
of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin
production. For example, the homozygous deletion of the antibody heavy-chain
joining
region (Jii) gene in chimeric and germ-line mutant mice results in complete
inhibition of
endogenous antibody production. Transfer of the human germ-line immunoglobulin
gene
array in such germ-line mutant mice will result in the production of human
antibodies upon
antigen challenge. The humanized or fully human antibodies may be prepared
according to
the methods described in U.S. Patent Nos. 5,770,429; 5,833,985; 5,837,243;
5,922,845;
6,017,517; 6,096,311; 6,111,166; 6,270,765; 6,303,755; 6,365,116; 6,410,690;
6,682,928;
and 6,984,720, the contents each of which are herein incorporated by
reference.
e. Anti-GFAP antibodies
102731 Anti-GFAP antibodies may be generated using the techniques described
above as
well as using routine techniques known in the art. In some embodiments, the
anti-GFAP
antibody may be an unconjugated GFAP antibody, such as GFAP antibodies
available from
Dako (Catalog Number: M0761), ThermoFisher Scientific (Catalog Numbers: MA5-
12023,
A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-
0670 1 , or MA1-20035), AbCam (Catalog Numbers: ab10062, ab4648, ab68428,
ab33922,
ab207165, ab190288, ab115898, or ab21837), EMD Millipore (Catalog Numbers:
FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628), Santa Cruz (Catalog
Numbers: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-
71143, sc-
65343, or sc-33673), Sigma-Aldrich (Catalog Numbers: G3893 or G6171) or Sino
Biological
Inc. (Catalog Number: 100140-R012-50). The anti-GFAP antibody may be
conjugated to a
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fluorophore, such as conjugated GFAP antibodies available from ThermoFisher
Scientific
(Catalog Numbers: A-21295 or A-21294), EMD Millipore (Catalog Numbers:
MAB3402X,
MA 133402B, B3402B, or Nil A B34.02C:3) or AbC am (Catalog
Numbers: ab49874 or
ab194325).
8. Variations on Methods
[0274] The disclosed methods of determining the presence or amount of analyte
of interest
(UCH-L1 and/or GFAP) present in a sample may be as described herein. The
methods may
also be adapted in view of other methods for analyzing analytes. Examples of
well-known
variations include, but are not limited to, immunoassay, such as sandwich
immunoassay (e.g.,
monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich
immunoassays, including enzyme detection (enzyme immunoassay (EIA) or enzyme-
linked
immunosorbent assay (ELISA), competitive inhibition immunoassay (e.g., forward
and
reverse), enzyme multiplied immunoassay technique (EMIT), a competitive
binding assay,
bioluminescence resonance energy transfer (BRET), one-step antibody detection
assay,
homogeneous assay, heterogeneous assay, capture on the fly assay, etc.
a. Immunoassay
[0275] The analyte of interest, and/or peptides of fragments thereof (e.g.,
UCH-L1 and/or
GFAP, and/or peptides or fragments thereof, i.e., UCH-L1 and/or GFAP
fragments), may be
analyzed using UCH-L1 and/or GFAP antibodies in an immunoassay. The presence
or
amount of analyte (e.g., UCH-L1 and/or GFAP) can be determined using
antibodies and
detecting specific binding to the analyte (e.g., UCH-L1 and/or GFAP). For
example, the
antibody, or antibody fragment thereof, may specifically bind to the analyte
(e.g., UCH-L1
and/or GFAP). If desired, one or more of the antibodies can be used in
combination with one
or more commercially available monoclonal/polyclonal antibodies. Such
antibodies are
available from companies such as R&D Systems, Inc. (Minneapolis, MN) and Enzo
Life
Sciences International, Inc. (Plymouth Meeting, PA).
[0276] The presence or amount of analyte (e.g., UCH-L1 and/or GFAP) present in
a body
sample may be readily determined using an immunoassay, such as sandwich
immunoassay
(e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal
sandwich
immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and
enzyme
detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay
(ELISA)
(e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, MN)). An example of
a point-
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of-care device that can be used is i-STATO (Abbott, Laboratories, Abbott Park,
IL). Other
methods that can be used include a chemiluminescent microparticle immunoassay,
in
particular one employing the ARCHITECT automated analyzer (Abbott
Laboratories,
Abbott Park, IL), as an example. Other methods include, for example, mass
spectrometry,
and immunohistochemistry (e.g., with sections from tissue biopsies), using
anti-analyte (e.g.,
anti-UCH-L1 and/or anti-GFAP) antibodies (monoclonal, polyclonal, chimeric,
humanized,
human, etc.) or antibody fragments thereof against analyte (e.g., UCH-L1
and/or GFAP).
Other methods of detection include those described in, for example, U.S.
Patent Nos.
6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615;
5,885,527;
5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is
hereby
incorporated by reference in its entirety. Specific immunological binding of
the antibody to
the analyte (e.g., UCH-L and/or GFAP) can be detected via direct labels, such
as fluorescent
or luminescent tags, metals and radionuclides attached to the antibody or via
indirect labels,
such as alkaline phosphatase or horseradish peroxidase.
[0277] The use of immobilized antibodies or antibody fragments thereof may be
incorporated into the immunoassay. The antibodies may be immobilized onto a
variety of
supports, such as magnetic or chromatographic matrix particles, the surface of
an assay plate
(such as microtiter wells), pieces of a solid substrate material, and the
like. An assay strip
can be prepared by coating the antibody or plurality of antibodies in an array
on a solid
support. This strip can then be dipped into the test sample and processed
quickly through
washes and detection steps to generate a measurable signal, such as a colored
spot.
102781 A homogeneous format may be used. For example, after the test sample is
obtained
from a subject, a mixture is prepared. The mixture contains the test sample
being assessed
for analyte (e.g., UCH-L1 and/or GFAP), a first specific binding partner, and
a second
specific binding partner. The order in which the test sample, the first
specific binding
partner, and the second specific binding partner are added to form the mixture
is not critical.
The test sample is simultaneously contacted with the first specific binding
partner and the
second specific binding partner. In some embodiments, the first specific
binding partner and
any UCH-L1 and/or GFAP contained in the test sample may form a first specific
binding
partner-analyte (e.g., UCH-L1 and/or GFAP)-antigen complex and the second
specific
binding partner may form a first specific binding partner-analyte of interest
(e.g., UCH-L1
and/or GFAP)-second specific binding partner complex. In some embodiments, the
second
specific binding partner and any UCH-L1 and/or GFAP contained in the test
sample may
form a second specific binding partner-analyte (e.g., UCH-L1)-antigen complex
and the first
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specific binding partner may form a first specific binding partner-analyte of
interest (e.g.,
UCH-L1 and/or GFAP)-second specific binding partner complex. The first
specific binding
partner may be an anti-analyte antibody (e.g., anti-UCH-L1 antibody that binds
to an epitope
having an amino acid sequence comprising at least three contiguous (3) amino
acids of SEQ
ID NO: 1 or anti-GFAP antibody that binds to an epitope having an amino acid
sequence
comprising at least three contiguous (3) amino acids of SEQ ID NO: 2). The
second specific
binding partner may be an anti-analyte antibody (e.g., anti-UCH-L1 antibody
that binds to an
epitope having an amino acid sequence comprising at least three contiguous (3)
amino acids
of SEQ ID NO: 1 or anti-GFAP antibody that binds to an epitope having an amino
acid
sequence comprising at least three contiguous (3) amino acids of SEQ ID NO:
2). Moreover,
the second specific binding partner is labeled with or contains a detectable
label as described
above.
102791 A heterogeneous format may be used. For example, after the test sample
is obtained
from a subject, a first mixture is prepared. The mixture contains the test
sample being
assessed for analyte (e.g., UCH-L1 and/or GFAP) and a first specific binding
partner,
wherein the first specific binding partner and any UCH-L1 and/or GFAP
contained in the test
sample form a first specific binding partner-analyte (e.g., UCH-L1 and/or
GFAP)-antigen
complex. The first specific binding partner may be an anti-analyte antibody
(e.g., anti-UCH-
Li antibody that binds to an epitope having an amino acid sequence comprising
at least three
contiguous (3) amino acids of SEQ Ill NO: 1 or anti-GI-AP antibody that binds
to an epitope
having an amino acid sequence comprising at least three contiguous (3) amino
acids of SEQ
ID NO: 2). The order in which the test sample and the first specific binding
partner are added
to form the mixture is not critical.
102801 The first specific binding partner may be immobilized on a solid phase.
The solid
phase used in the immunoassay (for the first specific binding partner and,
optionally, the
second specific binding partner) can be any solid phase known in the art, such
as, but not
limited to, a magnetic particle, a bead, a test tube, a microtiter plate, a cu
vette, a membrane, a
scaffolding molecule, a film, a filter paper, a disc, and a chip. In those
embodiments where
the solid phase is a bead, the bead may be a magnetic bead or a magnetic
particle. Magnetic
beads/particles may be ferromagnetic, ferrimagnetic, paramagnetic,
superparamagnetic or
ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy,
Cr0/, MnAs,
MnBi, Eu0, and NiO/Fe. Examples of ferrimagnetic materials include NiFe204,
CoFe204,
Fe304 tor Fe0-Fe203). Beads can have a solid core portion that is magnetic and
is surrounded
by one or more non-magnetic layers. Alternately, the magnetic portion can be a
layer around
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a non-magnetic core. The solid support on which the first specific binding
member is
immobilized may be stored in dry form or in a liquid. The magnetic beads may
be subjected
to a magnetic field prior to or after contacting with the sample with a
magnetic bead on which
the first specific binding member is immobilized.
[0281] After the mixture containing the first specific binding partner-analyte
(e.g., UCH-L1
or GFAP) antigen complex is formed, any unbound analyte (e.g., UCH-L1 and/or
GFAP) is
removed from the complex using any technique known in the art. For example,
the unbound
analyte (e.g., UCH-LI and/or GFAP) can be removed by washing. Desirably,
however, the
first specific binding partner is present in excess of any analyte (e.g., UCH-
L1 and/or GFAP)
present in the test sample, such that all analyte (e.g., UCH-L1 and/or GFAP)
that is present in
the test sample is bound by the first specific binding partner.
[0282] After any unbound analyte (e.g., UCH-L1 and/or GFAP) is removed, a
second
specific binding partner is added to the mixture to form a first specific
binding partner-
analyte of interest (e.g., UCH-L1 and/or GFAP)-second specific binding partner
complex.
The second specific binding partner may be an anti-analyte antibody (e.g.,
anti-UCH-L1
antibody that binds to an epitope having an amino acid sequence comprising at
least three
contiguous (3) amino acids of SEQ ID NO: 1 or anti-GFAP antibody that binds to
an epitope
having an amino acid sequence comprising at least three contiguous (3) amino
acids of SEQ
ID NO: 2). Moreover, the second specific binding partner is labeled with or
contains a
detectable label as described above.
[0283] The use of immobilized antibodies or antibody fragments thereof may be
incorporated into the immunoassay. The antibodies may be immobilized onto a
variety of
supports, such as magnetic or chromatographic matrix particles (such as a
magnetic bead),
latex particles or modified surface latex particles, polymer or polymer film,
plastic or plastic
film, planar substrate, the surface of an assay plate (such as microtiter
wells), pieces of a solid
substrate material, and the like. An assay strip can be prepared by coating
the antibody or
plurality of antibodies in an array on a solid support. This strip can then be
dipped into the
test sample and processed quickly through washes and detection steps to
generate a
measurable signal, such as a colored spot.
[0284] In some aspects, it is possible that other antibodies can be selected
which similarly
may assist with maintaining the dynamic range and low end sensitivity of the
immunoassays.
For example, it may be useful to select at least one first antibody (such as a
capture antibody
or first specific binding partner) that binds to an epitope near the N-
terminus of the 38 kDa
BDP and at least one second antibody (such as a detection antibody or second
specific
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binding partner) that binds to an epitope near the middle of the 38 kDa BDP,
e.g., near the
middle of the 38 kDa BDP, and that does not overlap with the first antibody.
Other variations
are possible and could be readily tested by one of ordinary skill, such as by
confirming
antibodies bind to different epitopes by examining binding to short peptides,
and then
screening antibody pairs using low calibrator concentration. Moreover,
selecting antibodies
of differing affinity for GFAP also can assist with maintaining or increasing
the dynamic
range of the assay. GFAP antibodies have been described in the literature and
are
commercially available.
(1) Sandwich immunoassay
[0285] A sandwich immunoassay measures the amount of antigen between two
layers of
antibodies (i.e., at least one capture antibody) and a detection antibody (L
e., at least one
detection antibody). The capture antibody and the detection antibody bind to
different
epitopes on the antigen, e.g., analyte of interest such as UCH-L1 and/or GFAP.
Desirably,
binding of the capture antibody to an epitope does not interfere with binding
of the detection
antibody to an epitope. Either monoclonal or polyclonal antibodies may be used
as the
capture and detection antibodies in the sandwich immunoassay.
[0286] Generally, at least two antibodies are employed to separate and
quantify analyte
(e.g., UCH-L1 and/or GFAP) in a test sample. More specifically, the at least
two antibodies
bind to certain epitopes of analyte (e.g., UCH-L1 and/or GFAP) forming an
immune complex
which is referred to as a "sandwich". One or more antibodies can be used to
capture the
analyte (e.g., UCH-L1 and/or GFAP) in the test sample (these antibodies are
frequently
referred to as a "capture" antibody or "capture" antibodies) and one or more
antibodies is
used to bind a detectable (namely, quantifiable) label to the sandwich (these
antibodies are
frequently referred to as the "detection" antibody or "detection" antibodies).
In a sandwich
assay, the binding of an antibody to its epitope desirably is not diminished
by the binding of
any other antibody in the assay to its respective epitope. Antibodies are
selected so that the
one or more first antibodies brought into contact with a test sample suspected
of containing
analyte (e.g., UCH-L1 and/or GFAP) do not bind to all or part of an epitope
recognized by
the second or subsequent antibodies, thereby interfering with the ability of
the one or more
second detection antibodies to bind to the analyte (e.g., UCH-L1 and/or GFAP).
[0287] The antibodies may be used as a first antibody in said immunoassay. The
antibody
immunospecifically binds to epitopes on analyte (e.g., UCH-L1 and/or (IMP). In
addition to
the antibodies of the present invention, said immunoassay may comprise a
second antibody
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that immunospecifically binds to epitopes that are not recognized or bound by
the first
antibody.
102881 A test sample suspected of containing analyte (e.g., UCH-L1 and/or
GFAP) can be
contacted with at least one first capture antibody (or antibodies) and at
least one second
detection antibodies either simultaneously or sequentially. In the sandwich
assay format, a
test sample suspected of containing analyte (e.g., UCH-L1 and/or GFAP) is
first brought into
contact with the at least one first capture antibody that specifically binds
to a particular
epitope under conditions which allow the formation of a first antibody-analyte
(e.g., UCH-LI
and/or GFAP) antigen complex. If more than one capture antibody is used, a
first multiple
capture antibody-UCH-L1 and/or GFAP antigen complex is formed. In a sandwich
assay, the
antibodies, preferably, the at least one capture antibody, are used in molar
excess amounts of
the maximum amount of analyte (e.g., UCH-L and/or GFAP) expected in the test
sample.
For example, from about 5 lag/mL to about 1 mg/mL of antibody per ml of
microparticle
coating buffer may be used.
LAnti-UCH-L1 Capture Antibody
102891 Optionally, prior to contacting the test sample with the at least one
first capture
antibody, the at least one first capture antibody can be bound to a solid
support which
facilitates the separation the first antibody-analyte (e.g., UCH-L1 and/or
GFAP) complex
from the test sample. Any solid support known in the art can be used,
including but not
limited to, solid supports made out of polymeric materials in the forms of
wells, tubes, or
beads (such as a microparticle). The antibody (or antibodies) can be bound to
the solid
support by adsorption, by covalent bonding using a chemical coupling agent or
by other
means known in the art, provided that such binding does not interfere with the
ability of the
antibody to bind analyte (e.g., UCH-L1 and/or GFAP). Moreover, if necessary,
the solid
support can be derivatized to allow reactivity with various functional groups
on the antibody.
Such derivatization requires the use of certain coupling agents such as, but
not limited to,
maleic anhydride, N-hydroxysuccinimide and 1-ethy1-3-(3-
dimethylaminopropyl)carbodiimide.
102901 After the test sample suspected of containing analyte (e.g., I JCH-1.1
and/or GFAP)
is incubated in order to allow for the formation of a first capture antibody
(or multiple
antibody)-analyte (e.g., UCH-L1 and/or GFAP) complex. The incubation can be
carried out
at a pH of from about 4.5 to about 10.0, at a temperature of from about 2 C to
about 45C,
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and for a period from at least about one (1) minute to about eighteen (18)
hours, from about
2-6 minutes, from about 7 -12 minutes, from about 5-15 minutes, or from about
3-4 minutes.
ii.Detection Antibody
[0291] After formation of the first/multiple capture antibody-analyte (e.g.,
UCH-L1 and/or
GFAP) complex, the complex is then contacted with at least one second
detection antibody
(under conditions that allow for the formation of a first/multiple antibody-
analyte (e.g., UCH-
Li and/or GFAP) antigen-second antibody complex). In some embodiments, the
test sample
is contacted with the detection antibody simultaneously with the capture
antibody. If the first
antibody-analyte (e.g., UCH-Li and/or GFAP) complex is contacted with more
than one
detection antibody, then a first/multiple capture antibody-analyte (e.g., UCH-
L1 and/or
GFAP)-multiple antibody detection complex is formed. As with first antibody,
when the at
least second (and subsequent) antibody is brought into contact with the first
antibody-analyte
(e.g., UCH-L1 and/or GFAP) complex, a period of incubation under conditions
similar to
those described above is required for the formation of the first/multiple
antibody-analyte
(e.g., UCH-L1 and/or GFAP)-second/multiple antibody complex. Preferably, at
least one
second antibody contains a detectable label. The detectable label can be bound
to the at least
one second antibody prior to, simultaneously with or after the formation of
the first/multiple
antibody-analyte (e.g., UCH-L1 and/or GFAP)-second/multiple antibody complex.
Any
detectable label known in the art can be used.
[0292] Chemiluminescent assays can be performed in accordance with the methods
described in Adamczyk et al., Anal. Chim. Acta 579(1): 61-67 (2006). While any
suitable
assay format can be used, a microplate chemiluminometer (Mithras LB-940,
Berthold
Technologies U.S.A., LLC, Oak Ridge, TN) enables the assay of multiple samples
of small
volumes rapidly. The chemiluminometer can be equipped with multiple reagent
injectors
using 96-well black polystyrene microplates (Costar #3792). Each sample can be
added into
a separate well, followed by the simultaneous/sequential addition of other
reagents as
determined by the type of assay employed. Desirably, the formation of
pseudobases in
neutral or basic solutions employing an acridinium aryl ester is avoided, such
as by
acidification. The chemiluminescent response is then recorded well-by-well. In
this regard,
the time for recording the chemiluminescent response will depend, in part, on
the delay
between the addition of the reagents and the particular acridinium employed.
[0293] The order in which the test sample and the specific binding partner(s)
are added to
form the mixture for chemiluminescent assay is not critical. If the first
specific binding
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partner is detectably labeled with an acridinium compound, detectably labeled
first specific
binding partner-antigen (e.g., UCH-L1 and/or GFAP) complexes form.
Alternatively, if a
second specific binding partner is used and the second specific binding
partner is detectably
labeled with an acridinium compound, detectably labeled first specific binding
partner-
analyte (e.g., UCH-L1 and/or GFAP)-second specific binding partner complexes
form. Any
unbound specific binding partner, whether labeled or unlabeled, can be removed
from the
mixture using any technique known in the art, such as washing.
[0294] Hydrogen peroxide can be generated in situ in the mixture or provided
or supplied to
the mixture before, simultaneously with, or after the addition of an above-
described
acridinium compound. Hydrogen peroxide can be generated in situ in a number of
ways such
as would be apparent to one skilled in the art.
[0295] Alternatively, a source of hydrogen peroxide can be simply added to the
mixture.
For example, the source of the hydrogen peroxide can be one or more buffers or
other
solutions that are known to contain hydrogen peroxide. In this regard, a
solution of hydrogen
peroxide can simply be added.
[0296] Upon the simultaneous or subsequent addition of at least one basic
solution to the
sample, a detectable signal, namely, a chemiluminescent signal, indicative of
the presence of
analyte (e.g., UCH-L1 and/or GFAP) is generated. The basic solution contains
at least one
base and has a pH greater than or equal to 10, preferably, greater than or
equal to 12.
Examples of basic solutions include, but are not limited to, sodium hydroxide,
potassium
hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium
carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and
calcium
bicarbonate. The amount of basic solution added to the sample depends on the
concentration
of the basic solution. Based on the concentration of the basic solution used,
one skilled in the
art can easily determine the amount of basic solution to add to the sample.
Other labels other
than chemiluminescent labels can be employed. For instance, enzymatic labels
(including but
not limited to alkaline phosphatase) can be employed.
[0297] The chemiluminescent signal, or other signal, that is generated can be
detected using
routine techniques known to those skilled in the art. Based on the intensity
of the signal
generated, the amount of analyte of interest (e.g., UCH-Li and/or GFAP) in the
sample can
be quantified. Specifically, the amount of analyte (e.g., UCH-L1 and/or GFAP)
in the sample
is proportional to the intensity of the signal generated. The amount of
analyte (e.g., UCH-Li
and/or GFAP) present can be quantified by comparing the amount of light
generated to a
standard curve for analyte (e.g., UCH-Li and/or GFAP) or by comparison to a
reference
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standard. The standard curve can be generated using serial dilutions or
solutions of known
concentrations of analyte (e.g., UCH-Li and/or GFAP) by mass spectroscopy,
gravimetric
methods, and other techniques known in the art.
(2) Forward Competitive Inhibition Assay
102981 In a forward competitive format, an aliquot of labeled analyte of
interest (e.g.,
analyte (e.g., UCH-L1 and/or GFAP) having a fluorescent label, a tag attached
with a
cleavable linker, etc.) of a known concentration is used to compete with
analyte of interest
(e.g., UCH-L1 and/or GFAP) in a test sample for binding to analyte of interest
antibody (e.g.,
UCH-L1 and/or GFAP antibody).
[0299] In a forward competition assay, an immobilized specific binding partner
(such as an
antibody) can either be sequentially or simultaneously contacted with the test
sample and a
labeled analyte of interest, analyte of interest fragment or analyte of
interest variant thereof.
The analyte of interest peptide, analyte of interest fragment or analyte of
interest variant can
be labeled with any detectable label, including a detectable label comprised
of tag attached
with a cleavable linker. In this assay, the antibody can be immobilized on to
a solid support.
Alternatively, the antibody can be coupled to an antibody, such as an
antispecies antibody,
that has been immobilized on a solid support, such as a microparticle or
planar substrate.
[0300] The labeled analyte of interest, the test sample and the antibody are
incubated under
conditions similar to those described above in connection with the sandwich
assay format.
Two different species of antibody-analyte of interest complexes may then be
generated.
Specifically, one of the antibody-analyte of interest complexes generated
contains a
detectable label (e.g., a fluorescent label, etc.) while the other antibody-
analyte of interest
complex does not contain a detectable label. The antibody-analyte of interest
complex can be,
but does not have to be, separated from the remainder of the test sample prior
to
quantification of the detectable label. Regardless of whether the antibody-
analyte of interest
complex is separated from the remainder of the test sample, the amount of
detectable label in
the antibody-analyte of interest complex is then quantified. The concentration
of analyte of
interest (such as membrane-associated analyte of interest, soluble analyte of
interest,
fragments of soluble analyte of interest, variants of analyte of interest
(membrane-associated
or soluble analyte of interest) or any combinations thereof) in the test
sample can then be
determined, e.g., as described above.
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(3) Reverse Competitive Inhibition Assay
[0301] In a reverse competition assay, an immobilized analyte of interest
(e.g., UCH-Li
and/or GFAP) can either be sequentially or simultaneously contacted with a
test sample and
at least one labeled antibody.
[0302] The analyte of interest can be bound to a solid support, such as the
solid supports
discussed above in connection with the sandwich assay format.
[0303] The immobilized analyte of interest, test sample and at least one
labeled antibody
are incubated under conditions similar to those described above in connection
with the
sandwich assay format. Two different species analyte of interest-antibody
complexes are
then generated. Specifically, one of the analyte of interest-antibody
complexes generated is
immobilized and contains a detectable label (e.g., a fluorescent label, etc.)
while the other
analyte of interest-antibody complex is not immobilized and contains a
detectable label. The
non-immobilized analyte of interest-antibody complex and the remainder of the
test sample
are removed from the presence of the immobilized analyte of interest-antibody
complex
through techniques known in the art, such as washing. Once the non-immobilized
analyte of
interest antibody complex is removed, the amount of detectable label in the
immobilized
analyte of interest-antibody complex is then quantified following cleavage of
the tag. The
concentration of analyte of interest in the test sample can then he determined
by comparing
the quantity of detectable label as described above.
(4) One-Step Immunoassay or "Capture on the Fly" Assay
[0304] In a capture on the fly immunoassay, a solid substrate is pre-coated
with an
immobilization agent. The capture agent, the analyte (e.g., UCH-L1 and/or
GFAP) and the
detection agent are added to the solid substrate together, followed by a wash
step prior to
detection. The capture agent can bind the analyte (e.g., UCH-L1 and/or GFAP)
and
comprises a ligand for an immobilization agent. The capture agent and the
detection agents
may be antibodies or any other moiety capable of capture or detection as
described herein or
known in the art. The ligand may comprise a peptide tag and an immobilization
agent may
comprise an anti-peptide tag antibody. Alternately, the ligand and the
immobilization agent
may be any pair of agents capable of binding together so as to be employed for
a capture on
the fly assay (e.g., specific binding pair, and others such as are known in
the art). More than
one analyte may be measured. In some embodiments, the solid substrate may be
coated with
an antigen and the analyte to be analyzed is an antibody.
[0305] In certain other embodiments, in a one-step immunoassay or "capture on
the fly", a
solid support (such as a microparticle) pre-coated with an immobilization
agent (such as
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biotin, streptavidin, etc.) and at least a first specific binding member and a
second specific
binding member (which function as capture and detection reagents,
respectively) are used.
The first specific binding member comprises a ligand for the immobilization
agent (for
example, if the immobilization agent on the solid support is streptavidin, the
ligand on the
first specific binding member may be biotin) and also binds to the analyte of
interest (e.g.,
UCH-L1 and/or GFAP). The second specific binding member comprises a detectable
label
and binds to an analyte of interest (e.g., UCH-L1 and/or GFAP). The solid
support and the
first and second specific binding members may be added to a test sample
(either sequentially
or simultaneously). The ligand on the first specific binding member binds to
the
immobilization agent on the solid support to form a solid support/first
specific binding
member complex. Any analyte of interest present in the sample binds to the
solid
support/first specific binding member complex to form a solid support/first
specific binding
member/analyte complex. The second specific binding member binds to the solid
support/first specific binding member/analyte complex and the detectable label
is detected.
An optional wash step may be employed before the detection. In certain
embodiments, in a
one-step assay more than one analyte may be measured. In certain other
embodiments, more
than two specific binding members can be employed. In certain other
embodiments, multiple
detectable labels can be added. In certain other embodiments, multiple
analytes of interest
can be detected, or their amounts, levels or concentrations, measured,
determined or assessed.
10306] 'the use of a capture on the fly assay can be done in a variety of
formats as described
herein, and known in the art. For example, the format can be a sandwich assay
such as
described above, but alternately can be a competition assay, can employ a
single specific
binding member, or use other variations such as are known.
9. Other Factors
103071 The methods of diagnosing, prognosticating, and/or assessing, as
described above,
can further include using other factors for the diagnosis, prognostication,
and assessment. In
some embodiments, traumatic brain injury may be diagnosed using the Glasgow
Coma Scale.
Other tests, scales or indices can also be used either alone or in combination
with the
Glasgow Coma Scale. An example is the Ranchos Los Amigos Scale. The Ranchos
Los
Amigos Scale measures the levels of awareness, cognition, behavior and
interaction with the
environment. The Ranchos Los Amigos Scale includes: Level I: No Response;
Level II:
Generalized Response; Level III: Localized Response; Level IV: Confused-
agitated; Level V:
Confused-inappropriate; Level VI: Confused-appropriate; Level VII: Automatic-
appropriate;
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and Level VIII: Purposeful-appropriate. Another example is the Rivermead Post-
Concussion
Symptoms Questionairre, a self-report scale to measure the severity of post-
concussive
symptoms following TB!. Patients are asked to rate how severe each of 16
symptoms (e.g.,
headache, dizziness, nausea, vomiting) has been over the past 24 hours. In
each case, the
symptom is compared with how severe it was before the injury occurred
(premorbid). These
symptoms are reported by severity on a scale from 0 to 4: not experienced, no
more of a
problem, mild problem, moderate problem, and severe problem.
10. Samples
103081 In some embodiments, the sample is obtained after the subject, such as
a human
subject, sustained an injury to the head caused by physical shaking, blunt
impact by an
external mechanical or other force that results in a closed or open head
trauma, one or more
falls, explosions or blasts or other types of blunt force trauma. In some
embodiments, the
sample is obtained after the subject, such as a human subject, has ingested or
been exposed to
a fire, chemical, toxin or combination of a fire, chemical and toxin. Examples
of such
chemicals and/or toxins include, molds, asbestos, pesticides and insecticides,
organic
solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and
cyanide),
organic metals (such as methyl mercury, tetraethyl lead and organic tin)
and/or one or more
drugs of abuse. In some embodiments, the sample is obtained from a subject,
such as a
human subject, that suffers from an autoimmune disease, a metabolic disorder,
a brain tumor,
hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial
infection,
meningitis, hydrocephalus, or any combinations thereof_
103091 In yet another embodiment, the methods described herein use samples
that also can
be used to determine whether or not a subject has or is at risk of developing
a TBI (such as a
mild TBI, moderate TBI, severe TBI, or moderate to severe TBI) by determining
the levels of
UCH-L1 and/or GFAP in a subject using the anti-UCH-L1 and/or anti-GFAP
antibodies
described below, or antibody fragments thereof. Thus, in particular
embodiments, the
disclosure also provides a method for determining whether a subject having, or
at risk for,
traumatic brain injuries, discussed herein and known in the art, is a
candidate for therapy or
treatment. Generally, the subject is at least one who: (i) has experienced an
injury to the
head; (ii) ingested and/or been exposed to one or more chemicals and/or
toxins; (iii) suffers
from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a
viral infection
(e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis,
hydrocephalus, or
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any combinations thereof; or (iv) any combinations of (i)-(iii); or, who has
actually been
diagnosed as having, or being at risk for TBI (such as, for example, subjects
suffering from
an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral
infection (e.g.,
SARS-CoV-2), a fungal infection, a bacterial infection, meningitis,
hydrocephalus, or any
combinations thereof), and/or who demonstrates an unfavorable (i.e.,
clinically undesirable)
concentration or amount of UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragment,
as
described herein.
a. Test or Biological Sample
[0310] As used herein, "sample", "test sample", "biological sample" refer to
fluid sample
containing or suspected of containing UCH-L1 and/or GFAP. The sample may be
derived
from any suitable source. In some cases, the sample may comprise a liquid,
fluent particulate
solid, or fluid suspension of solid particles. In some cases, the sample may
be processed prior
to the analysis described herein. For example, the sample may be separated or
purified from
its source prior to analysis; however, in certain embodiments, an unprocessed
sample
containing UCH-L1 and/or GFAP may be assayed directly. In a particular
example, the
source of UCH-L1 and/or GFAP is a human bodily substance (e.g., bodily fluid,
blood such
as whole blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus,
lacrimal fluid,
lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal
fluid, oropharyngeal
specimen, nasopharyngeal specimen, feces, tissue, organ, or the like). Tissues
may include,
but are not limited to skeletal muscle tissue, liver tissue, lung tissue,
kidney tissue,
myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc. The
sample may be a
liquid sample or a liquid extract of a solid sample. In certain cases, the
source of the sample
may be an organ or tissue, such as a biopsy sample, which may be solubilized
by tissue
disintegration/cell lysis.
[0311] A wide range of volumes of the fluid sample may be analyzed. In a few
exemplary
embodiments, the sample volume may be about 0.5 nL, about 1 nL, about 3 nL,
about 0.01
uL, about 0.1 uL, about 1 uL, about 5 uL, about 10 uL, about 100 .tL, about 1
mL, about 5
mL, about 10 mL, or the like. In some cases, the volume of the fluid sample is
between about
0.01 p,L and about 10 mL, between about 0.01 p,L and about 1 mL, between about
0.01 p,L
and about 100 [IL, or between about 0.1 [IL and about 10 pt.
[0312] In some cases, the fluid sample may be diluted prior to use in an
assay. For example,
in embodiments where the source of UCH-L1 and/or GFAP is a human body fluid
(e.g.,
blood, serum), the fluid may be diluted with an appropriate solvent (e.g., a
buffer such as
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PBS buffer). A fluid sample may be diluted about 1-fold, about 2-fold, about 3-
fold, about 4-
fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,
prior to use. In
other cases, the fluid sample is not diluted prior to use in an assay.
[0313] In some cases, the sample may undergo pre-analytical processing. Pre-
analytical
processing may offer additional functionality such as nonspecific protein
removal and/or
effective yet cheaply implementable mixing functionality. General methods of
pre-analytical
processing may include the use of electrokinetic trapping, AC electrokinetics,
surface
acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other
pre-
concentration techniques known in the art. In some cases, the fluid sample may
be
concentrated prior to use in an assay. For example, in embodiments where the
source of
UCH-Li and/or GFAP is a human body fluid (e.g., blood, serum), the fluid may
be
concentrated by precipitation, evaporation, filtration, centrifugation, or a
combination thereof.
A fluid sample may be concentrated about 1-fold, about 2-fold, about 3-fold,
about 4-fold,
about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior
to use.
b. Controls
103141 It may be desirable to include a control sample. The control sample may
be
analyzed concurrently with the sample from the subject as described above. The
results
obtained from the subject sample can be compared to the results obtained from
the control
sample. Standard curves may be provided, with which assay results for the
sample may be
compared. Such standard curves present levels of marker as a function of assay
units, i.e.,
fluorescent signal intensity, if a fluorescent label is used. Using samples
taken from multiple
donors, standard curves can be provided for reference levels of the UCH-L1
and/or GFAP in
normal healthy tissue, as well as for "at-risk- levels of the UCH-L1 and/or
GFAP in tissue
taken from donors, who may have one or more of the characteristics set forth
above.
[0315] Thus, in view of the above, a method for determining the presence,
amount, or
concentration of UCH-L1 and/or GFAP in a test sample is provided. The method
comprises
assaying the test sample for UCH-Li and/or GFAP by an immunoassay, for
example,
employing at least one capture antibody that binds to an epitope on UCH-L1
and/or GFAP
and at least one detection antibody that binds to an epitope on UCH-Li and/or
GFAP which
is different from the epitope for the capture antibody and optionally includes
a detectable
label, and comprising comparing a signal generated by the detectable label as
a direct or
indirect indication of the presence, amount or concentration of UCH-LI and/or
GFAP in the
test sample to a signal generated as a direct or indirect indication of the
presence, amount or
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concentration of UCH-L1 and/or GFAP in a calibrator. The calibrator is
optionally, and is
preferably, part of a series of calibrators in which each of the calibrators
differs from the
other calibrators in the series by the concentration of UCH-L1 and/or GFAP.
11. Kit
10316] Provided herein is a kit, which may be used for assaying or assessing a
test sample
for UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragment. The kit comprises at
least one
component for assaying the test sample for UCH-L1 and/or GFAP instructions tor
assaying
the test sample for UCH-L1 and/or GFAP. For example, the kit can comprise
instructions for
assaying the test sample for UCH-L1 and/or GFAP by immunoassay, e.g.,
chemiluminescent
microparticle immunoassay. Instructions included in kits can be affixed to
packaging
material or can be included as a package insert. While the instructions are
typically written
or printed materials, they are not limited to such. Any medium capable of
storing such
instructions and communicating them to an end user is contemplated by this
disclosure. Such
media include, but are not limited to, electronic storage media (e.g.,
magnetic discs, tapes,
cartridges, chips), optical media (e.g., CD ROM), and the like. As used
herein, the term
"instructions" can include the address of an internet site that provides the
instructions.
103171 The at least one component may include at least one composition
comprising one or
more isolated antibodies or antibody fragments thereof that specifically bind
to UCH-L1
and/or GFAP. The antibody may be a UCH-L1 and/or GFAP capture antibody and/or
a
UCH-L1 and/or GFAP detection antibody.
103181 Alternatively or additionally, the kit can comprise a calibrator or
control, e.g.,
purified, and optionally lyophilized, UCH-L1 and/or GFAP, and/or at least one
container
(e.g., tube, microtiter plates or strips, which can be already coated with an
anti-UCH-L1
and/or GFAP monoclonal antibody) for conducting the assay, and/or a buffer,
such as an
assay buffer or a wash buffer, either one of which can be provided as a
concentrated solution,
a substrate solution for the detectable label (e.g., an enzymatic label), or a
stop solution.
Preferably, the kit comprises all components, i.e., reagents, standards,
buffers, diluents, etc.,
which are necessary to perform the assay. The instructions also can include
instructions for
generating a standard curve.
103191 The kit may further comprise reference standards for quantifying UCH-L1
and/or
GFAP. The reference standards may be employed to establish standard curves for
interpolation and/or extrapolation of UCH-L1 and/or GFAP concentrations. The
reference
standards may include a high UCH-L1 and/or GFAP concentration level, for
example, about
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100000 pg/mL, about 125000 pg/mL, about 150000 pg/mL, about 175000 pg/mL,
about
200000 pg/mL, about 225000 pg/mL, about 250000 pg/mL, about 275000 pg/mL, or
about
300000 pg/mL; a medium UCH-L1 and/or GFAP concentration level, for example,
about
25000 pg/mL, about 40000 pg/mL, about 45000 pg/mL, about 50000 pg/mL, about
55000
pg/mL, about 60000 pg/mL, about 75000 pg/mL or about 100000 pg/mL; and/or a
low UCH-
Li and/or GFAP concentration level, for example, about 1 pg/mL, about 5 pg/mL,
about 10
pg/mL, about 12.5 pg/mL, about 15 pg/mL, about 20 pg/mL, about 25 pg/mL, about
30
pg/mL, about 35 pg/mL, about 40 pg/mL, about 45 pg/mL, about 50 pg/mL, about
55 pg/mL,
about 60 pg/mL, about 65 pg/mL, about 70 pg/mL, about 75 pg/mL, about 80
pg/mL, about
85 pg/mL, about 90 pg/mL, about 95 pg/mL, or about 100 pg/mL.
[0320] Any antibodies, which are provided in the kit, such as recombinant
antibodies
specific for UCH-L1 and/or GFAP, can incorporate a detectable label, such as a
fluorophore,
radioactive moiety, enzyme, biotin/avidin label, chromophore, chemiluminescent
label, or the
like, or the kit can include reagents for labeling the antibodies or reagents
for detecting the
antibodies (e.g., detection antibodies) and/or for labeling the analytes
(e.g., UCH-L1 and/or
GFAP) or reagents for detecting the analyte (e.g., UCH-L1 and/or GFAP). The
antibodies,
calibrators, and/or controls can be provided in separate containers or pre-
dispensed into an
appropriate assay format, for example, into microtiter plates,
[0321] Optionally, the kit includes quality control components (for example,
sensitivity
panels, calibrators, and positive controls). Preparation of quality control
reagents is well-
known in the art and is described on insert sheets for a variety of
immunodiagnostic products.
Sensitivity panel members optionally are used to establish assay performance
characteristics,
and further optionally are useful indicators of the integrity of the
immunoassay kit reagents,
and the standardization of assays,
[0322] The kit can also optionally include other reagents required to conduct
a diagnostic
assay or facilitate quality control evaluations, such as buffers, salts,
enzymes, enzyme co-
factors, substrates, detection reagents, and the like. Other components, such
as buffers and
solutions for the isolation and/or treatment of a test sample (e.g.,
pretreatment reagents), also
can be included in the kit. The kit can additionally include one or more other
controls. One
or more of the components of the kit can be lyophilized, in which case the kit
can further
comprise reagents suitable for the reconstitution of the lyophilized
components.
[0323] The various components of the kit optionally are provided in suitable
containers as
necessary, e.g., a microtiter plate. The kit can further include containers
for holding or
storing a sample (e.g., a container or cartridge for a urine, whole blood,
plasma, or serum
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sample). Where appropriate, the kit optionally also can contain reaction
vessels, mixing
vessels, and other components that facilitate the preparation of reagents or
the test sample.
The kit can also include one or more instrument for assisting with obtaining a
test sample,
such as a syringe, pipette, forceps, measured spoon, or the like.
[0324] If the detectable label is at least one acridinium compound, the kit
can comprise at
least one acridinium-9-carboxamide, at least one acridinium-9-carboxylate aryl
ester, or any
combination thereof. If the detectable label is at least one acridinium
compound, the kit also
can comprise a source of hydrogen peroxide, such as a buffer, solution, and/or
at least one
basic solution. If desired, the kit can contain a solid phase, such as a
magnetic particle, bead,
test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film,
filter paper, disc, or
chip.
[0325] If desired, the kit can further comprise one or more components, alone
or in further
combination with instructions, for assaying the test sample for another
analyte, which can be
a biomarker, such as a biomarker of traumatic brain injury or disorder.
a. Adaptation of Kit and Method
103261 The kit (or components thereof), as well as the method for assessing or
determining
the concentration of UCH-L1 and/or GFAP in a test sample by an immunoassay as
described
herein, can be adapted for use in a variety of automated and semi-automated
systems
(including those wherein the solid phase comprises a microparticle), as
described, e.g., U.S.
Patent No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881,
2004/0018577, 2005/0054078, and 2006/0160164 and as commercially marketed
e.g., by
Abbott Laboratories (Abbott Park, IL) as Abbott Point of Care (i-STATO or i-
STAT Alinity,
Abbott Laboratories) as well as those described in U.S. Patent Nos. 5,089,424
and 5,006,309,
and as commercially marketed, e.g., by Abbott Laboratories (Abbott Park, IL)
as
ARCHITECT or the series of Abbott Alinity devices.
[0327] Some of the differences between an automated or semi-automated system
as
compared to a non-automated system (e.g., ELISA) include the substrate to
which the first
specific binding partner (e.g., analyte antibody or capture antibody) is
attached (which can
affect sandwich formation and analyte reactivity), and the length and timing
of the capture,
detection, and/or any optional wash steps. Whereas a non-automated format such
as an
ELISA may require a relatively longer incubation time with sample and capture
reagent (e.g.,
about 2 hours), an automated or semi-automated format (e.g., ARCHITECT and
any
successor platform, Abbott Laboratories) may have a relatively shorter
incubation time (e.g.,
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approximately 18 minutes for ARCHITECT ). Similarly, whereas a non-automated
format
such as an ELIS A may incubate a detection antibody such as the conjugate
reagent for a
relatively longer incubation time (e.g., about 2 hours), an automated or semi-
automated
format (e.g., ARCHITECT and any successor platform) may have a relatively
shorter
incubation time (e.g., approximately 4 minutes for the ARCHITECT and any
successor
platform).
[0328] Other platforms available from Abbott Laboratories include, but are not
limited to,
AxSYMO, IMx (see, e.g., U.S. Patent No. 5,294,404, which is hereby
incorporated by
reference in its entirety), PRISM , EIA (bead), and QuantumTM II, as well as
other
platforms. Additionally, the assays, kits, and kit components can be employed
in other
formats, for example, on electrochemical or other hand-held or point-of-care
assay systems.
As mentioned previously, the present disclosure is, for example, applicable to
the commercial
Abbott Point of Care (i-STATO, Abbott Laboratories) electrochemical
immunoassay system
that performs sandwich immunoassays. Immunosensors and their methods of
manufacture
and operation in single-use test devices are described, for example in, U.S.
Patent No.
5,063,081, U.S. Patent App. Publication Nos. 2003/0170881, 2004/0018577,
2005/0054078,
and 2006/0160164, which are incorporated in their entireties by reference for
their teachings
regarding same.
[0329] Regarding the adaptation of an assay to the i-STAT system, the
following
configuration is preferred. A microfabricated silicon chip is manufactured
with a pair of gold
amperometric working electrodes and a silver-silver chloride reference
electrode. On one of
the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized
capture
antibody are adhered to a polymer coating of patterned polyvinyl alcohol over
the electrode.
This chip is assembled into all i-STATO cartridge with a fluidics format
suitable for
immunoassay. On a portion of the silicon chip, there is a specific binding
partner for UCH-
Li and/or GFAP, such as one or more UCH-L1 and/or GFAP antibodies (one or more
monoclonal/polyclonal antibody or a fragment thereof, a variant thereof, or a
fragment of a
variant thereof that can bind UCH-L1 and/or GFAP) or one or more anti-UCH-L1
and/or
GFAP DVD-Igs (or a fragment thereof, a variant thereof, or a fragment of a
variant thereof
that can bind UCH-L1 and/or GFAP), either of which can be detectably labeled.
Within the
fluid pouch of the cartridge is an aqueous reagent that includes p-aminophenol
phosphate.
[0330] In operation, a sample from a subject suspected of suffering from TBI
is added to
the holding chamber of the test cartridge, and the cartridge is inserted into
the i-STATO
reader. A pump element within the cartridge pushes the sample into a conduit
containing the
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chip. The sample is brought into contact with the sensors allowing the enzyme
conjugate to
dissolve into the sample. The sample is oscillated across the sensors to
promote formation of
the sandwich of approximately 2-12 minutes. In the penultimate step of the
assay, the sample
is pushed into a waste chamber and wash fluid, containing a substrate for the
alkaline
phosphatase enzyme, is used to wash excess enzyme conjugate and sample off the
sensor
chip. In the final step of the assay, the alkaline phosphatase label reacts
with p-aminophenol
phosphate to cleave the phosphate group and permit the liberated p-aminophenol
to be
electrochemically oxidized at the working electrode. Based on the measured
current, the
reader is able to calculate the amount of UCH-L1 and/or GFAP in the sample by
means of an
embedded algorithm and factory-determined calibration curve.
[0331] The automated and semi-automated systems described herein for use in
the methods
of the present disclosure can utilize one or more computer programs, software
or algorithms
to provide the determination (readout) of whether to perform an CT scan (e.g.,
based on a
positive result) or not to perform an CT scan (e.g., based on a negative
result). For example,
the computer program(s) or software (e.g., making use of an algorithm) can
provide an
interpretation (regardless of whether one, two or more samples are used) that:
(1) when the
level of GFAP and UCH-L1 is less than the reference level (or cutoff) that the
result is
negative meaning that no CT scan will be performed; or (2) when the level or
GFAP and/or
UCH-L1 is greater than or equal to the reference level (or cutoff) that the
result is positive
meaning that an Cl scan will be performed. '[he computer program(s) or
software can
provide other appropriate interpretations, such as, whether the reference
level is or is not
correlated with a positive head CT, the presence of an intracranial lesion or
with control
subjects that have not suffered a traumatic brain injury, whether the subject
suffering from
the TBI should be monitored and/or treated with a TBI treatment, etc. Such
computer
programs or software are well known in the art.
10332] The methods and kits as described herein necessarily encompass other
reagents and
methods for carrying out the immunoassay. For instance, encompassed are
various buffers
such as are known in the art and/or which can be readily prepared or optimized
to be
employed, e.g., for washing, as a conjugate diluent, and/or as a calibrator
diluent. An
exemplary conjugate diluent is ARCHITECT conjugate diluent employed in
certain kits
(Abbott Laboratories, Abbott Park, IL) and containing 2-(N-
morpholino)ethanesulfonic acid
(MES), a salt, a protein blocker, an antimicrobial agent, and a detergent. An
exemplary
calibrator diluent is ARCHITECT human calibrator diluent employed in certain
kits
(Abbott Laboratories, Abbott Park, IL), which comprises a buffer containing
MES, other salt,
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a protein blocker, and an antimicrobial agent. Additionally, as described in
U.S. Patent
Application No. 61/142,048 filed December 31, 2008, improved signal generation
may be
obtained, e.g., in an i-S TAT cartridge format, using a nucleic acid sequence
linked to the
signal antibody as a signal amplifier.
[0333] While certain embodiments herein are advantageous when employed to
assess
disease, such as traumatic brain injury, the assays and kits also optionally
can be employed to
assess UCH-L1 and/or GFAP in other diseases, disorders, and conditions as
appropriate.
[0334] The method of assay also can be used to identify a compound that
ameliorates
diseases, such as traumatic brain injury. For example, a cell that expresses
UCH-L1 and/or
GFAP can be contacted with a candidate compound. The level of expression of
UCH-L1
and/or GFAP in the cell contacted with the compound can be compared to that in
a control
cell using the method of assay described herein.
[0335] The present invention has multiple aspects, illustrated by the
following non-limiting
examples.
EXAMPLES
Example 1
i-STATO UCH-L1 Assay
[0336] The i-STAT UCH-L1 assay was used in a TBI patient population study of
subjects
having a negative CT scan. Monoclonal antibody pairs, such as Antibody A as a
capture
monoclonal antibody and Antibody B and C as a detection monoclonal antibody,
were used.
Antibody A is an exemplary anti-UCH-L1 antibody that was internally developed
at Abbott
Laboratories (Abbott Park, IL). Antibody B and C recognize different epitopes
of UCH-L1
and enhance the detection of antigen in the sample that were developed by
Banyan
Biomarkers (Alachua, Florida). Other antibodies that were internally developed
at Abbott
Laboratories (Abbott Park, IL), or other commercially available antibodies,
also show or are
expected to show similar enhancement of signal when used together as capture
antibodies or
detection antibodies, in various combinations. The UCH-L1 assay design was
evaluated
against key performance attributes. The cartridge configuration was Antibody
Configuration:
Antibody A (Capture Antibody)/Antibody B+C (Detection Antibody); Reagent
conditions:
0.8% solids, 125 vis /mL Fab Alkaline Phosphatase cluster conjugate; and
Sample Inlet Print:
UCII-L1 standard. The assay time was 10-15 min (with 7-12 min sample capture
time).
Example 2
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i-STATO GFAP Assay
[0337] The i-STATO GFAP assay was used in a TBI patient population study.
Monoclonal
antibody pairs, such as Antibody A as a capture monoclonal antibody and
Antibody B as a
detection monoclonal antibody, were used. Antibody A and Antibody B are
exemplary anti-
GFAP antibodies that were internally developed at Abbott Laboratories (Abbott
Park, IL).
Antibody A and Antibody B bind to epitopes within the same GFAP breakdown
product. The
GFAP assay design was evaluated against key performance attributes. The
cartridge
configuration was Antibody Configuration: Antibody A (Capture
Antibody)/Antibody B
(Detection Antibody); Reagent conditions: 0.8% solids, 250 litg/mL Fab
Alkaline
Phosphatase cluster conjugate; and Sample Inlet Print: GFAP specific. The
assay time was
10-15 min (with 7-12 min sample capture time).
Example 3
Assessment of GFAP and UCH-L1 Levels in Subjects with Negative CT scan
[0338] To evaluate blood levels of biomarkers such as GFAP and UCH-L1 in
predicting
computed tomography (CT)-negative TBI, blood samples were collected from adult
(i.e., 18
years of age or more) trauma subjects who underwent a head CT scan. Biomarker
concentrations of subjects with negative (Marshall classification =1) CT scans
were
evaluated.
[0339] FIGS. 1A-D show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
Glascow Coma Score (GCS) severity for those subjects having CT scans negative
for TBI.
Samples were assessed within 12 hours from injury (FIG. 1A, 1C) or within
about 24 hours
(i.e., within about 24.1 hours) from injury (FIG. 1B, 1D). GFAP levels are
shown in FIG.
lA and FIG. 1B. UCH-L1 levels are shown in FIG. 1C and FIG. 1D. At 12 hours,
the
reference level for GFAP was between about 99 pg/mL and about 1674 pg/mL and
at 24
hours, between about 114 pg/mL and about 941 pg/mL. At 12 hours, the reference
level for
UCH-L1 was between about 229 pg/mL and about 665 pg/mL and at 24 hours,
between about
162 pg/mL and about 314 pg/mL.
[0340] FIGS. 2A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
loss of consciousness after injury for those subjects having CT scans negative
for TBI.
Samples were assessed within 4 hours (FIG. 2A, 2D), within 12 hours (FIG. 2B,
2E), or
within about 24 hours (i.e., within about 24.1 hours) from injury (FIG. 2C,
2F). GFAP levels
are shown in FIG. 2A-2C. UCH-L1 levels are shown in FIGS. 2D-F.
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[0341] FIGS. 3A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
MRI results for those subjects having CT scans negative for TBI. Samples were
assessed
within 4 hours (FIG. 3A, 3D), within 12 hours (FIG. 3B, 3E) or within about 24
hours (i.e.,
within about 24.1 hours) from injury (FIG. 3C, 3F). GFAP levels are shown in
FIGS. 3A-3C.
UCH-L1 levels are shown in FIGS. 3D-F. At about 4 hours, the reference level
for GFAP
was about 50 pg/mL to about 220 pg/mL, at 12 hours, the reference level for
GFAP was
between about 76 pg/mL and about 326 pg/mL and at 24 hours, between about 76
pg/mL and
about 383 pg/mL. At 4 hours, the reference level for UCH-Li was about 235
pg/mL to about
396 pg/mL, at 12 hours, the reference level for UCH-L1 was between about 210
pg/mL and
about 285 pg/mL and at 24 hours, between about 150 pg/mL and about 169 pg/mL.
[0342] FIGS. 4A-F show ROC analysis of UCH-L1 levels or GFAP levels correlated
with
post-traumatic amnesia (present vs. absent) for those subjects having CT scans
negative for
TBI. Samples were assessed within 4 hours (FIG. 4A, 4D), within 12 hours (FIG.
4B, 4E) or
within about 24 hours (i.e., within about 24.1 hours) from injury (FIG. 4C,
4F). GFAP levels
are shown in FIGS. 4A-C. UCH-L1 levels are shown in FIGS. 4D-F.
[0343] It will be readily apparent to those skilled in the art that other
suitable modifications
and adaptations of the methods of the present disclosure described herein are
readily
applicable and appreciable, and may be made using suitable equivalents without
departing
from the scope of the present disclosure or the aspects and embodiments
disclosed
herein. Having now described the present disclosure in detail, the same will
be more clearly
understood by reference to the following examples, which are merely intended
only to
illustrate some aspects and embodiments of the disclosure, and should not be
viewed as
limiting to the scope of the disclosure. The disclosures of all journal
references, U.S. patents,
and publications referred to herein are hereby incorporated by reference in
their entireties.
[0344] The present invention has multiple aspects, illustrated by the non-
limiting examples
described herein.
[0345] It is understood that the foregoing detailed description and
accompanying examples
are merely illustrative and are not to be taken as limitations upon the scope
of the invention,
which is defined solely by the appended claims and their equivalents.
[0346] Various changes and modifications to the disclosed embodiments will be
apparent to
those skilled in the art. Such changes and modifications, including without
limitation those
relating to the chemical structures, substituents, derivatives, intermediates,
syntheses,
compositions, formulations, or methods of use of the invention, may be made
without
departing from the spirit and scope thereof.
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[0347] For reasons of completeness, various aspects of the invention are set
out in the
following numbered clauses:
[0348] Clause 1. In an improvement of a method for aiding in the diagnosis and
evaluation
of a subject that has sustained or may have sustained an injury to the head,
the method
comprising performing, simultaneously or sequentially: (1) an assay on a
sample obtained
from the subject within about 24 hours after an actual or suspected injury to
the head to
measure or detect a level of a biomarker in the sample, said biomarker
comprising ubiquitin
carboxy-terminal hydrolase LI (UCH-L1), glial fibrillary acidic protein
(GFAP), or a
combination thereof; and (2) a head computerized tomography scan on the
subject, within a
clinically-relevant time frame, wherein the improvement comprises diagnosing
the subject as
more likely than not as having traumatic brain injury (TBI) if the level of
the biomarker is
higher than a reference level and the head CT scan is negative for a TBI.
[0349] Clause 2. The improvement of clause 1, further comprising treating the
subject for a
TBI if the level of the biomarker is higher than a reference level and the
imaging procedure is
negative for a TBI.
[0350] Clause 3. The improvement of any of clause 1 or clause 2, wherein the
reference
level is correlated with a cutoff level associated with: (a) levels in
subjects that have
sustained a head injury; (b) the occurrence of TBI in a subject; (c) stage of
TBI in a subject
such as mild, moderate, severe, or moderate to severe; (d) loss of
consciousness in a subject;
(e) MRI positive for TBI rather than negative; (f) the occurrence of amnesia
in a subject (i.e.,
amnesia present vs. absent) or (g) severity of TBI in a subject.
[0351] Clause 4. The improvement of any of clauses 1-3, wherein the sample is
taken
within about 0 to about 12 hours after the actual or suspected injury to the
head or within
about 12 to about 24 hours after the suspected injury to the head.
[0352] Clause 5. The improvement of any of clauses 1-4, wherein measuring the
level of
UCH-L1 is done by an immunoassay or a clinical chemistry assay.
[0353] Clause 6. The improvement of any of clauses 1-5, wherein measuring the
level of
GFAP is done by immunoassay or a clinical chemistry assay.
[0354] Clause 7. The improvement of any of clauses 1-6, wherein the assay is
performed
using a point-of-care assay or single molecule detection.
[0355] Clause 8. The improvement of any of clauses 1-7, wherein the sample is
selected
from the group consisting of a blood sample, a urine sample, a cerebrospinal
fluid sample, a
tissue sample, a bodily fluid sample, a saliva sample, an oropharyngeal
specimen, and a
nasopharyngeal specimen.
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[0356] Clause 9. The improvement of any of clauses 1-8, wherein the sample is
obtained
after the subject has sustained or may have sustained an injury to the head
caused by physical
shaking, blunt impact by an external mechanical or other force that results in
a closed or open
head trauma, one or more falls, explosions or blasts or other types of blunt
force trauma.
[0357] Clause 10. The improvement of any of clauses 1-9, wherein the sample is
obtained
after the subject has ingested or been exposed to a fire, chemical, toxin or
combination of a
fire, chemical and toxin.
[0358] Clause 11. The improvement of clause 10, wherein the chemical or toxin
is mold,
asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a
gas, an organic
metal, a drug of abuse or one or more combinations thereof.
[0359] Clause 12. The improvement of any of clauses 1-11, wherein the sample
is obtained
from a subject that suffers from an autoimmune disease, a metabolic disorder,
a brain tumor,
hypoxia, a viral infection, a fungal infection, a bacterial infection,
meningitis, hydrocephalus,
or any combinations thereof.
[0360] Clause 13. The improvement of any of clauses 1-12, wherein said method
can be
carried out on any subject without regard to factors selected from the group
consisting of the
subject's clinical condition, the subject's laboratory values, the subject's
classification as
suffering from mild, moderate, severe, or moderate to severe traumatic brain
injury, the
subject's exhibition of low, moderate or high levels of UCH-Li, GFAP, or UCH-
L1 and
GPAP, and the timing of any event wherein said subject has sustained or may
have sustained
an injury to the head.
[0361] Clause 14. The improvement of any of clauses 1-13, further comprising
monitoring
the subject.
[0362] Clause 15. A method for aiding in the diagnosis and evaluation of a
subject that has
sustained or may have sustained an injury to the head, the method comprising:
[0363] a. performing, simultaneously or sequentially: (1) an assay on a sample
obtained
from the subject within about 24 hours after an actual or a suspected injury
to the head to
measure or detect a level of a biomarker in the sample, said biomarker
comprising ubiquitin
carboxy-terminal hydrolase Li (UCH-L1), glial fibrillary acidic protein
(GFAP), or a
combination thereof; and (2) a head computerized tomography (CT) scan, within
a clinically-
relevant time frame; and
[0364] b. diagnosing the subject as more likely than not as having traumatic
brain injury
(TBI) if the level of the biomarker is higher than a reference level and the
head CT scan is
negative for a TBI.
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[0365] Clause 16. The method of clause 15, further comprising treating the
subject for a
TBI if the level of the biomarker is higher than a reference level and the CT
scan is negative
for a TB!.
[0366] Clause 17. The method of any of clause 15 or clause 16, wherein the
reference level
is correlated with a cutoff level associated with: (a) levels in subjects that
have sustained a
head injury; (b) the occurrence of TBI in a subject; (c) stage of TBI in a
subject such as mild,
moderate, severe, or moderate to severe; (d) loss of consciousness in a
subject; (e) MRI
positive for TBI rather than negative; (f) the occurrence of amnesia in a
subject (i.e., amnesia
present vs. absent) or (g) severity of TBI in a subject.
[0367] Clause 18. The method of any of clauses 15-17, wherein the sample is
taken within
about 0 to about 12 hours after the actual or suspected injury to the head or
within about 12 to
about 24 hours after the suspected injury to the head.
[0368] Clause 19. The method of any of clauses 15-18, wherein measuring the
level of
UCH-L1 is done by an immunoassay or a clinical chemistry assay.
[0369] Clause 20. The method of any of clauses 15-19, wherein measuring the
level of
GFAP is done by immunoassay or a clinical chemistry assay.
103701 Clause 21. The method of any of clauses 15-20, wherein the assay is
performed
using a point-of-care assay or single molecule detection.
[0371] Clause 22. The method of any of clauses 15-21, wherein the sample is
selected from
the group consisting of a blood sample, a urine sample, a cerebrospinal fluid
sample, a tissue
sample, a bodily fluid sample, a saliva sample, an oropharyngeal specimen, and
a
nasopharyngeal specimen.
[0372] Clause 23. The method of any of clauses 15-22, wherein the sample is
obtained
after the subject has sustained or may have sustained an injury to the head
caused by physical
shaking, blunt impact by an external mechanical or other force that results in
a closed or open
head trauma, one or more falls, explosions or blasts or other types of blunt
force trauma.
[0373] Clause 24. The method of any of clauses 15-22, wherein the sample is
obtained
after the subject has ingested or been exposed to a fire, chemical, toxin or
combination of a
fire, chemical and toxin.
[0374] Clause 25. The method of any of clause 24, wherein the chemical or
toxin is mold,
asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a
gas, an organic
metal, a drug of abuse or one or more combinations thereof.
[0375] Clause 26. The method of any of clauses 15-22, wherein the sample is
obtained
from a subject that suffers from an autoimmune disease, a metabolic disorder,
a brain tumor,
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hypoxia, a viral infection, a fungal infection, a bacterial infection,
meningitis, hydrocephalus,
or any combinations thereof.
[0376] Clause 27. The method of any of clauses 15-22, wherein said method can
be carried
out on any subject without regard to factors selected from the group
consisting of the
subject's clinical condition, the subject's laboratory values, the subject's
classification as
suffering from mild, moderate, severe, or severe to moderate to severe
traumatic brain injury,
the subject's exhibition of low, moderate or high levels of UCH-L1, GFAP or
UCH-L1 and
GFAP, and the timing of any event wherein said subject has sustained or may
have sustained
an injury to the head.
[0377] Clause 28. The method of any of clauses 15-27, further comprising
monitoring the
subject.
[0378] Clause 29. In an improvement of a method for aiding in the diagnosis
and
evaluation of a subject that has sustained or may have sustained an injury to
the head,
wherein the subject received a head computerized tomography (CT) scan negative
for
traumatic brain injury (TBI) within a clinically-relevant time frame of the
actual or suspected
head injury, the method comprising performing, simultaneously or sequentially
with the head
CT an assay on a sample obtained from the subject within about 24 hours after
the actual or
suspected head injury to measure or detect a level of a biomarker in the
sample, said
biomarker comprising ubiquitin carboxy-terminal hydrolase Li (UCH-L1), glial
fibrillary
acidic protein (GFAP), or a combination thereof; wherein the improvement
comprises
diagnosing the subject as more likely than not as having traumatic brain
injury (TBI) if the
level of the biomarker is higher than a reference level.
[0379] Clause 30. In an improvement of a method for aiding in the diagnosis
and
evaluation of a subject that has sustained or may have sustained an injury to
the head, the
method comprising performing an assay on a sample obtained from the subject
within about
24 hours after an actual or suspected injury to the head to measure or detect
a level of a
biomarker in the sample, said biomarker comprising ubiquitin carboxy-terminal
hydrolase Li
(UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof;
and wherein the
improvement comprises diagnosing the subject as more likely than not as having
traumatic
brain injury (TBI) if the level of the biomarker is higher than a reference
level, and either a
head computerized tomography (CT) scan on the subject within a clinically-
relevant time
frame is negative for a TBI, or no head CT scan is performed on the subject.
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[0380] Clause 31. The improvement of clause 30, further comprising treating
the subject
for a 'TBI if the level of the biomarker is higher than a reference level and
optionally, if
performed, a head CT scan is negative for a TB!.
[0381] Clause 32. The improvement of any of clause 30 or clause 31, wherein
the
reference level is correlated with a cutoff level associated with: (a) levels
in subjects that
have sustained a head injury; (b) the occurrence of TBI in a subject; (c)
stage of TBI in a
subject such as mild, moderate, severe, or moderate to severe; (d) loss of
consciousness in a
subject; (e) MRI positive for TBI rather than negative; (f) the occurrence of
amnesia in a
subject (i.e., amnesia present vs. absent) or (g) severity of TBI in a
subject.
[0382] Clause 33. The improvement of any of clauses 30-32, wherein the sample
is taken
within about 0 to about 12 hours after the actual or suspected injury to the
head or within
about 12 to about 24 hours after the actual or suspected injury to the head.
[0383] Clause 34. The improvement of any of clauses 30-33, wherein measuring
the level
of UCH-L1 is done by an immunoassay or a clinical chemistry assay.
[0384] Clause 35. The improvement of any of clauses 30-34, wherein measuring
the level
of GFAP is done by immunoassay or a clinical chemistry assay.
103851 Clause 36. The improvement of any of clauses 30-35, wherein the assay
is
performed using a point-of-care assay or single molecule detection.
[0386] Clause 37. The improvement of any of clauses 30-36, wherein the sample
is
selected from the group consisting of a blood sample, a urine sample, a
cerebrospinal fluid
sample, a tissue sample, a bodily fluid sample, a saliva sample, an
oropharyngeal specimen,
and a nasopharyngeal specimen
[0387] Clause 38. The improvement of any of clauses 30-37, wherein the sample
is
obtained after the subject sustained an actual injury to the head caused by
physical shaking,
blunt impact by an external mechanical or other force that results in a closed
or open head
trauma, one or more falls, explosions or blasts or other types of blunt force
trauma.
[0388] Clause 39. The improvement of any of clauses 30-37, wherein the sample
is
obtained after the subject has ingested or been exposed to a fire, chemical,
toxin or
combination of a fire, chemical and toxin.
[0389] Clause 40. The improvement of clause 39, wherein the chemical or toxin
is mold,
asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a
gas, an organic
metal, a drug of abuse or one or more combinations thereof.
[0390] Clause 4L The improvement of any of clauses 30-37, wherein the sample
is
obtained from a subject that suffers from an autoimmune disease, a metabolic
disorder, a
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brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial
infection, meningitis,
hydrocephalus, or any combinations thereof.
[0391] Clause 42. The improvement of any of clauses 30-41, wherein said method
can be
carried out on any subject without regard to factors selected from the group
consisting of the
subject's clinical condition, the subject's laboratory values, the subject's
classification as
suffering from mild, moderate, severe, or severe to moderate to severe
traumatic brain injury,
the subject's exhibition of low, moderate or high levels of UCH-L1, and the
timing of any
event wherein said subject has sustained or may have sustained an injury to
the head.
[0392] Clause 43. The improvement of any of clauses 30-42, further comprising
monitoring the subject.
[0393] Clause 44. The improvement of any of clauses 1-14, wherein the blood
sample is a
whole blood sample, a serum sample, or a plasma sample.
[0394] Clause 45. The method of any of clauses 1-14 or 44, wherein the subject
is a human
subject.
[0395] Clause 46. The method of any of clauses 15-28, wherein the blood sample
is a
whole blood sample, a serum sample, or a plasma sample.
103961 Clause 47. The method of any of clauses 15-28 or 46, wherein the
subject is a
human subject.
[0397] Clause 48. The improvement of any of clauses 29-42, wherein the blood
sample is a
whole blood sample, a serum sample, or a plasma sample.
[0398] Clause 49. The improvement of any of clauses 29-42 or 48, wherein the
subject is a
human subject.
[0399] Clause 50. The improvement of any of clauses 29-49, wherein the
reference level
for GFAP is from about 30 pg/mL to about 1700 pg/mL and the reference level
for UCH-L1
is from about 150 pg/mL to about 700 pg/mL.
[0400] Clause 51. The improvement of clause 50, wherein the reference level
for GFAP
from about 90 pg/mL to about 1680 pg/mL and the reference level for UCH-L1 is
from about
220 pg/mL to about 670 pg/mL.
[0401] Clause 52. The improvement of clause 51, wherein the reference level
for GFAP is
from about 110 pg/mL to about 950 pg/mL and the reference level for UCH-L1 is
from about
160 pg/mL to about 320 pg/mL.
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