Note: Descriptions are shown in the official language in which they were submitted.
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S10013 AND ISOFOR1VIS
FOR
DETECTION OF NEUROLOGICAL CONDITIONS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This claims priority from U.S. provisional Application No.
62/455,787, filed
February 7, 2017, which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions of matter and
antibodies for the
detection of biomarkers. More particularly, the present invention provides for
synthetic
compositions of matter in addition to methods, processes, kits and in vitro
diagnostic devices to
assist with biomarker identification.
BACKGROUND OF THE INVENTION
[0003] S100 calcium-binding protein B (S10013) is a protein of the S-
100 protein family.
S100 proteins are localized in the cytoplasm and nucleus of a wide range of
cells and are
involved in the regulation of a number of cellular processes such as cell
cycle progression and
differentiation. Due to its prevalence in astrocytes, S10013 has been heavily
studied and
identified as an important marker for diagnosing or predicting injuries and
conditions of the
central nervous system (CNS) including traumatic brain injury, neoplasia,
Alzheimer's disease,
Down's syndrome, epilepsy, and amyotrophic lateral sclerosis. However, it has
been found that
S10013 is expressed in several non-neuronal tissues, including adipose,
skeletal muscle, cardiac,
chondrocytes and epidermal cells. Therefore, these non-neuronal sources of
S10013 cannot be
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excluded as sources of elevation, thus precluding a clinical validation of an
S1000 diagnostic
assay for any condition.
[0004] While its biological function is still somewhat unclear,
intracellularly, 51000 is
involved in regulation of variety of cell activities and in regulation of cell
morphology
(Goncalves et al., 2008, Kleindienst & Bullock 2006). It has been noted,
however, that when
released into extracellular space, 51000 seems to have both toxic/degenerative
and
trophic/reparative roles depending on the concentration (Goncalves et al.,
2008).
[0005] Because of this abundance in many cell types, it is often
difficult to use 51000 as a
biomarker of any condition due to its lack of specificity. For this reason,
51000 historically fails
as a viable biomarker for many conditions which occur in a multi-trauma
setting. For example,
51000 is also present in human melanocytes, is a reliable marker for melanoma
malignancy both
in bioptic tissue and in serum. In addition, 51000 has further been predictive
of non-neuronal
traumas, such as liver injury, femoral fracture, and myocardial infarction, in
addition to soft
tissue injuries, bone damage, and other diseases causing organ injury such as
sepsis. Thus, the
reliability of 51000 as a measure of any particular condition depends upon the
type of injury and
the specificity of the assay.
[0006] Notwithstanding, because of its abundance, S1000 tends to be a
very sensitive
biomarker, and is often still looked at as a potential candidate for
diagnosing many different
types of injuries and disorders. One such injury that 51000 has been found to
help diagnose is
injuries to the CNS. For example, elevated SlOOB levels have been found to
reflect presence of
neuropathological conditions TBI or neurodegenerative diseases. Its potential
clinical use is
substantiated by standard modalities for prognosticating the extent of CNS
damage: alterations in
neuroimaging, cerebrospinal pressure, and other brain molecular markers (NSE
and GFAP).
[0007] Several factors exist which make 51000 a prime candidate for CNS
detection. First,
it has been found that normal 51000 levels in patients reliably exclude major
CNS pathology. In
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addition, levels have been reported to rise prior to detectable changes in
intracerebral pressure,
neuroimaging, and neurological examination findings. It has further been found
that S1000
levels are elevated before seizures suggesting blood-brain barrier (BBB)
leakage may be early
event in seizure development. An important application for the use of S1000 as
a biomarker is
selection of patients with minor head injury who need no further
neuroradiological evaluation.
Studies comparing CT scans and S1000 levels demonstrate S1000 values below
0.12 ng/mL are
associated with low risk neuroradiological changes (e.g. intracranial
hemorrhage or brain
swelling) or significant clinical sequelae.
[0008] One problem identified is the lack of specificity of S1000
because if its prevalence.
Originally considered specific to astroglia in the CNS, S1000 presents in
other extracerebral
tissues such as adipocytes, chondrocytes and bone marrow cells (Donato 2001).
The general
accepted theory, then, is that S1000 is non-specific to the CNS, despite many
factors indicating
that it is a prime candidate for detecting CNS pathologies. As a result, it is
believed, that certain
isoforms exist of S1000 which are specific to different organ types, that if
identified, may
differentially diagnose organ specific injuries, and thus providing a highly
specific S1000 assay.
Thus, there remains an unmet need for an injury specific S1000 assay that is
sensitive to a
particular organ injury even in a multi-trauma environment.
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions of matter, antibodies
and antigens
related thereto, methods, kits and in vitro diagnostic processes specifically
designed to detect
protein markers that are differentially present in the samples of patients
suffering from one of
several injury and/or disorders alone or in a multi-trauma environment. The
inventive
compositions of matter are those isolated nucleic acid sequence having 50%,
60%, 70%, 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ
ID NO. 7
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and SEQ ID NO. 8. These compositions of matter, and antibodies raised against
such
compositions of matter, assist with providing a sensitive, specific, quick,
and non-invasive
method to aid in diagnosis of a myriad of injuries and/or disorders that may
be present in a
subject, even in the event of a multi-trauma environment, by detecting and
determining the
amount of biomarkers that are indicative to the respective injury type. The
measurement of these
markers, alone or in combination with other markers for the injury type, in
patient samples
provides information that a diagnostician can correlate with a probable
diagnosis of the extent of
an injury.
[0010] The present invention further provides in vitro diagnostic
processes and kits for
.. detecting injuries and disorders in a subject for a particular injury, even
in the event that the
subject has suffered multiple traumas prior to the measurement of the
biomarkers.
[0011] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates a 2D gel detecting the isoforms of S1000 in
serum, showing the
capture of the isolated nucleic acid sequence having 50%, 60%, 70%, 80%, 85%,
90%, 95%,
96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7 and
SEQ ID NO.
8, which represent the S1000 isoforms, S10007-S10008, respectively.
[0013] FIG. 2 illustrates a method to generate antibodies to peptides
corresponding to the
isolated nucleic acid sequences of Exon 2, Exon 3 and Exon 4 of the S1000 cDNA
having 50%,
60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid
sequence of
SEQ ID NO. 7 and SEQ ID NO. 8.
[0014] FIG. 3 illustrates the Exon arrangements for the formation of
nucleic acid sequence
of SEQ ID NO. 7 and SEQ ID NO. 8. The isoforms so identified consist of Exon
1, Exon 2 and
either that portion of Exon 3 (S100B7) or Exon 4 (S100B8) that completes the
isoform sequence
to the stop codon (TGA, TAG nucleic acid sequence).
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[0015] FIG. 4 illustrates the various Exon locations in the complete
S100(3 mRNA. The
Exons include Exon 1, Exon 2 and then either Exon 3 to stop codon TGA or Exon
4 to stop
codon TGA or TAG.
5 DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention has utility in the diagnosis and
management of an injury or
abnormal condition in a subject, with the injury or condition being alone, or
in combination with
a series of other injuries or disorders. Through the measurement of biomarkers
in a biological
sample from a subject, such as the isolated nucleic acid sequence having 50%,
60%, 70%, 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of
S100(37 or
S100(38, alone or in combination with each other or in combination with one or
more additional
biomarkers known in the art, a determination of subject's injury or condition
is provided with
greater specificity than previously attainable, even in the event of a multi-
trauma patient. The
present description is directed toward a first isoform of S100(3, selected
from the isolated nucleic
acid sequence having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identity
to a nucleic acid sequence of S100(37 or S100(38, as a biomarker.
Surprisingly, by combining the
detection of more than one biomarker of S100(31 through S100(38, a synergistic
result is achieved
where multiple injuries may be simultaneously diagnosed and distinguished from
one another.
[0017] As used herein the term "diagnosing" means recognizing the
presence or absence of
a neurological or other condition such as an injury or disease. Diagnosing is
optionally referred
to as the result of an assay wherein a particular ratio or level of a
biomarker is detected or is
absent.
[0018] As used herein a "ratio" is either a positive ratio wherein the
level of the target is
greater than the target in a second sample or relative to a known or
recognized baseline level of
the same target. A negative ratio describes the level of the target as lower
than the target in a
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second sample or relative to a known or recognized baseline level of the same
target. A neutral
ratio describes no observed change in target biomarker.
[0019] As used herein an injury is an alteration in cellular or
molecular integrity, activity,
level, robustness, state, or other alteration that is traceable to an event.
Injury illustratively
includes a physical, mechanical, chemical, biological, functional, infectious,
or other modulator
of cellular or molecular characteristics. An event is illustratively, a
physical trauma such as an
impact (percussive) or a biological abnormality such as a stroke resulting
from either blockade or
leakage of a blood vessel. An event is optionally an infection by an
infectious agent. A person
of skill in the art recognizes numerous equivalent events that are encompassed
by the terms
injury or event. An injury is optionally a physical event such as a percussive
impact. Injury may
further include a disease or genetic abnormality such as Parkinson's Disease,
Alzheimer's
disease, ALS, etc.
[0020] It is to be understood that in instances where a range of values
are provided the range
is intended to encompass not only the endpoint values of the range but also
intermediate values
of the range as explicitly being included within the range and varying by the
last significant
figure of the range. By way of example, a recited range of from 1 to 4 is
intended to include 1-2,
1-3, 2-4, 3-4, and 1-4
General
[0021] The present invention provides compositions of matter,
antibodies and antigens
related thereto, methods, kits and in vitro diagnostic processes specifically
used to detect protein
markers that are differentially present in the samples of patients suffering
from one of several
injury and/or disorders alone or in a multi-trauma environment. The inventive
compositions of
matter are those identified as the isolated nucleic acid sequence having 50%,
60%, 70%, 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ
ID NO. 7
and SEQ ID NO. 8. These compositions of matter, and antibodies raised against
such
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compositions of matter, assist with providing a sensitive, specific, quick,
and non-invasive
method to aid in diagnosis of a myriad of injury and/or disorders that may be
present in a subject,
even in the event of a multi-trauma environment, by detecting and determining
the amount of
biomarkers that are indicative to the respective injury type. The measurement
of these markers,
alone or in combination of other markers for the injury type, in patient
samples provides
information that a diagnostician can correlate with a probable diagnosis of
the extent of an
injury.
Compositions ofMatter
[0022] Antibody-based assays are preferred for analyzing a biological
sample for the
presence of a biomarker and one or more other biomarkers of a particular
injury or condition.
Suitable western blotting methods are described below in the examples section.
For more rapid
analysis (as may be important in emergency medical situations), immunosorbent
assays (e.g.,
ELISA and RIA) and immunoprecipitation assays may be used.
[0023] Reagents as described herein may be any antibody to a protein or
peptide sequence
or any antigen to detect an antibody formed as part of an autoimmune response
in a subject. In
particular for the antigens used for these detection methods and devices, they
may be those
proteins or peptides generated to detect an antibody that has been produced by
a subject's own
tissues as an autoimmune response to cells, tissues or native proteins of the
organism in which it
was formed. Preferably these antigens are peptides having the following
sequence for each of
the defined isoforms from which antibodies are, have been, or will be
generated:
[0024] SEQ ID NO. 1 is the publicly recognized (LOCUS NP 006263 92;
CAG46920, aa
(molecular wt. about 10,713 Daltons) linear, DEFINITION protein 51000 [Homo
sapiens],
ACCESSION NP 006263 VERSION NP 006263.1 GI: 5454034 or GI: 4957424 DBSOURCE
REFSEQ: accession NM 006272.2; embl accession CR542123.1) generic sequence
commonly
referred to or linked to all S1000 studies currently recognized in the art.
Because of the recent
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discoveries of the several isoforms, not previously known in the art, the
conventional name
S10013 necessarily needs to be changed to S100131 as is the convention within
the scientific
research community. This peptide, or antibodies raised against this peptide,
is recognized in the
art as not being specific to any particular injury or disorder. This nucleic
acid sequence is known
to have the following amino acid sequence as noted is the result of splicing
Exon 1 and Exon 2:
MSELEKAMVALIDVFHQYSGREGDKHKLKK SELKELINNEL SHFLEEIKEQEVVDKVME
TLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE
[0025] SEQ ID NO 2 is the first of the compositions of matter
identified in U.S. Patent
Application S/N: 14/293,758 formed as a result of the splicing Exon 1, Exon 2
(truncated #1)
and the newly discovered Exon 3 of the human nucleic acid sequence, thus the
protein as a result
of the translation of the nucleic acid sequence is named S100(32. The protein
S100(32, found to
have a molecular weight of an estimated 11,295 Daltons, has been found to have
the following
amino acid sequence:
MSELEKAMVALIDVFHQYSGREGDKHKLKK SELKELINNEL SHFLEEIKEQEVVDKVME
TLDNDGDGECDFQEFMAFVAMRRSCKKAD SKGLQP SRS
[0026] SEQ ID NO 3 is the second of the compositions of matter
identified in U.S. Patent
Application S/N: 14/293,758 formed as a result of Exon 1 (truncated) and Exon
3 of the human
nucleic acid sequence, thus the protein as a result of the translation of the
nucleic acid sequence
is named S100(33. The peptide S100(33, found to have a molecular weight of
about 2,750
Daltons has been found to have the following amino acid sequence:
MSELEKAMRRSCKKADSKGLQPSRS
[0027] SEQ ID NO 4 is the third of the compositions of matter
identified in U.S. Patent
Application S/N: 14/293,758 formed as a result of Exon 1 (truncated) and Exon
2 of the human
nucleic acid sequence, thus the protein as a result of the translation of the
nucleic acid sequence
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is named S100(34. The peptide S100(34, found to have a molecular weight of
about 5,950
Daltons has been found to have the following amino acid sequence:
MSELEKAMEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE
[0028]
SEQ ID NO 5 is the fourth of the compositions of matter identified in U.S.
Patent
Application S/N: 14/293,758 formed as a result of Exon 1 (truncated), Exon 2
(truncated #1) and
Exon 3 of the human nucleic acid sequence, thus the protein as a result of the
translation of the
nucleic acid sequence is named S100(35. The peptide S100(35, found to have a
molecular weight
of about 6,500 Daltons has been found to have the following amino acid
sequence:
MSELEKAMEIKEQEVVDKVMETLDNDGDGECDF QEFMAFVAMRRSCKKAD SKGLQP S
RS
[0029]
SEQ ID NO 6: is the fifth of the compositions of matter identified in U.S.
Patent
Application S/N: 14/293,758 formed as a result of Exon 1, Exon 2 (truncated
#2) and Exon 3 of
the human nucleic acid sequence, thus the protein as a result of the
translation of the nucleic acid
sequence is named S100(36. The peptide S100(36, found to have a molecular
weight of ¨7,600
Daltons has been found to have the following amino acid sequence:
MSELEKAMVALIDVFHQYSGREGDKHKLKK SELKELINNEL SHFLEEIKEQERRSCKKA
DSKGLQPSRS
[0030]
SEQ ID NO 7: is the first of the several newly discovered compositions of
matter
formed as a result of Exon 1, a newly discovered Exon 2 and Exon 3b of the
human nucleic acid
sequence, thus the protein as a result of the translation of the nucleic acid
sequence is named
S100(37. The peptide 5100137 is found to have a molecular weight of ¨10,342.75
Daltons has
been found to have the following
sequence:
MSELEKAMVALIDVFHQYSGREGDKHKLKK SELKELINNEL SHFLERW SLPMLPRLVSN
FLCSSYPPALASQSAGITGNQRAGGCGQSHGNTGQ or a sequence which is at least 10%
identical to said sequence, or a portion of any said sequence.
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[0031] SEQ ID
NO 8: is the fifth of the several newly discovered compositions of matter
formed as a result of Exon 1, a newly discovered Exon 2 and Exon 4a of the
human nucleic acid
sequence, thus the protein as a result of the translation of the nucleic acid
sequence is named
S100(38. The peptide S100(38, found to have a molecular weight of ¨10,281.8
Daltons has been
5 found to have the following
sequence:
MSELEKAMVALIDVFHQYSGREGDKHKLKK SELKELINNEL SHFLERW SLPMLPRLVSN
FLCSSYPPALASQSAGITETVMQESRQQGLAA or a sequence which is at least 10% identical
to said sequence, or a portion of any said sequence.
[0032] It
should be appreciated that while the sequences described above are those
S10013
10 isoforms found for the human polypeptide sequence, that similar
isoforms may be available in
rat, mouse, rabbit, bovine, Chinese hamster, Rhesus monkey, zebrafish, goat,
chicken, dog,
domestic cat, and pig and other species having a similar sequence alignment to
the S10013 for
humans. These alternative sequences may be substituted in the inventive
processes, methods,
assays, and in vitro diagnostic devices described herein. In addition, while
discovery of these
proteins are a landmark discovery in diagnostics of injuries in a multi-trauma
patient, other
compositions of matter of importance are those antibodies raised against any
of the peptide
sequences represented by the nucleic acid sequence having 50%, 60%, 70%, 80%,
85%, 90%,
95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO's 7-
8, or
antibodies raised against the rat, mouse, rabbit, bovine, Chinese hamster,
Rhesus monkey,
zebrafish, goat, chicken, dog, domestic cat, and pig and other similar species
formulations of the
similar sequence alignment.
[0033] An
antibody is optionally labeled. A person of ordinary skill in the art
recognizes
numerous labels operable herein. Labels and labeling kits are commercially
available. Labels
illustratively include, fluorescent labels, biotin, peroxidase,
radionucleotides, or other label
known in the art. Alternatively, a detection species of another antibody or
other compound
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known to the art is used as form detection of a biomarker bound by an
antibody. If the antibody
is directly conjugated with a detectable label, such as an enzyme,
fluorophore, or radioisotope,
the presence of the label is optionally detected by examining the substrate
for the detectable
label. Alternatively, a detectably labeled secondary antibody that binds the
marker-specific
antibody is added to the substrate.
[0034] It should be appreciated that because of the poor antigenicity
for the peptide
sequences of the new Exon 2 and 3a and 4a, that a better method of generating
response of mice
to create antibodies was needed. As a result, in at least one embodiment, a
novel MAP (Multiple
Antigen Peptide) system as modified was used. In such embodiment, FIG. 2
presents the basis
for the novel and distinct modifications of the modified MAP method.
[0035] The Multiple Antigenic Peptide (MAP) dendrimer system has four
sections to
increase the induction of a stronger immune response, reduce enzymatic
degradation and
enhance molecular recognition to antigenic peptides for antibody generation.
The MAP core
which consists of a branched lysine core scaffold has a bare cell-penetrating
peptide attached to
one end and several copies of the peptide epitopes attached to the other. To
increase functionality
and immune stimulation lipophilic moieties are attached to the peptide
epitopes. The MAP
system is hydrophobic and requires formulation in an emulsion medium that is
biocompatible for
administration into the animals normally used to generate the antibodies. The
emulsion medium
required is one that does not oxidize the methionines, cysteines and
tryptophans in the peptide
sequences yet is able to handle their hydrophobic characteristics.
Biomarker Methods
[0036] An exemplary process for detecting the presence or absence of a
biomarker, alone or
in combination, in a biological sample involves obtaining a biological sample
from a subject,
such as a human, contacting the biological sample with a compound or an agent
capable of
detecting of the marker being analyzed, illustratively including an antibody
or aptamer, and
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analyzing binding of the compound or agent to the sample after washing. Those
samples having
specifically bound compound or agent express the marker being analyzed.
[0037] The present invention further provides a step of comparing the
quantity of one or
more biomarkers to normal levels of uninjured patients to determine the injury
or condition of
the subject. It is appreciated that selection of additional biomarkers allows
one to identify the
types of cells implicated in an abnormal organ or physical condition as well
as the nature of cell
death. The practice of an inventive process provides a test which can help a
physician determine
suitable therapeutics to administer for optimal benefit of the subject
suffering from a particular
condition in a multi-trauma scenario and to select a therapeutic that may be
administered in
combination with other therapeutics for the other measured injuries.
[0038] In vitro techniques for detection of a marker illustratively
include enzyme linked
immunosorbent assays (ELISAs), radioimmunoassay, radioassay, western blot,
Southern blot,
northern blot, immunoprecipitation, immunofluorescence, mass spectrometry, RT-
PCR, PCR,
liquid chromatography, high performance liquid chromatography, enzyme activity
assay, cellular
assay, positron emission tomography, mass spectroscopy, combinations thereof,
or other
technique known in the art. Furthermore, in vivo techniques for detection of a
marker include
introducing a labeled agent that specifically binds the marker into a
biological sample or test
subj ect.
[0039] Numerous permutations of these basic immunoassays are also
operative in the
invention. These include the biomarker-specific antibody, as opposed to the
sample being
immobilized on a substrate, and the substrate is contacted with a biomarker
conjugated with a
detectable label under conditions that cause binding of antibody to the
labeled marker. The
substrate is then contacted with a sample under conditions that allow binding
of the marker being
analyzed to the antibody. A reduction in the amount of detectable label on the
substrate after
washing indicates that the sample contained the marker.
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[0040] Although antibodies are preferred for use in the invention
because of their extensive
characterization, any other suitable agent (e.g., a peptide, an aptamer, or a
small organic
molecule) that specifically binds a biomarker is optionally used in place of
the antibody in the
above described immunoassays. For example, an aptamer that specifically binds
S100137 and/or
one or more of the other S10013 protein isoforms or peptides of the same might
be used.
Aptamers are nucleic acid-based molecules that bind specific ligands. Methods
for making
aptamers with a particular binding specificity are known as detailed in U.S.
Patent Nos.
5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985;
5,567,588;
5,683,867; 5,637,459; and 6,011,020.
KITS
[0041] In yet another aspect, the invention provides kits for aiding a
diagnosis of a
particular injury, degree of severity of injury, subcellular localization
and/or a particular
disorder, wherein the kits can be used to detect the markers of the present
invention. For
example, the kits can be used to detect any one or more of the markers
described herein, which
markers are differentially present in samples of a patient and normal
subjects. The kits of the
invention have many applications. For example, the kits can be used to
differentiate if a subject
has sepsis, bone injury, muscle or tissue injury, or brain injury. In another
example, the kits can
be used to identify compounds that modulate expression of one or more of the
markers in in vitro
or in vivo animal models to determine the effects of treatment.
[0042] In one embodiment, a kit comprises (a) an antibody that specifically
binds to a
marker (a capture agent); and (b) a detection agent for detecting the amount
of the marker. Such
kits can be prepared from the materials described above, and the previous
discussion regarding
the materials (e.g., antibodies, detection reagents, immobilized supports,
etc.) is fully applicable
to this section and will not be repeated. Optionally, the kit may further
comprise pre-
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fractionation spin columns. In one embodiment, the kit may further comprise
instructions for
suitable operation parameters in the form of a label or a separate insert.
[0043] In one embodiment, the invention includes a diagnostic kit for
use in screening
serum containing antigens of the peptide of the invention. These antigens may
be one of any of
the peptides represented by nucleic acid molecules having 50%, 60%, 70%, 80%,
85%, 90%,
95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence of SEQ ID NO. 7-
8. The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive with peptide
or polynucleotide antigens and means for detecting the binding of the
polynucleotide or peptide
antigen to the antibody. In one embodiment, the antibody is attached to a
solid support.
Antibodies used in the inventive kit are those raised against any one of the
peptides represented
by nucleic acid molecules having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or
99% SEQ ID NO's 7-8. In one embodiment, the antibody is a monoclonal or
polyclonal
antibody raised against the rat, rabbit or human forms of the isoforms of
S1000 described herein.
The detecting means of the kit includes a second, labeled monoclonal or
polyclonal antibody.
Alternatively, or in addition thereto, the detecting means includes a labeled,
competing antigen.
[0044] Optionally, the kit may further comprise a standard or control
information so that the
test sample can be compared with the control information standard to determine
if the test
amount of a marker detected in a sample is a diagnostic amount consistent with
a diagnosis of a
particular injury, degree of severity of the injury, subcellular localization,
a particular disorder
and/or effect of treatment on the patient.
[0045] In one embodiment, a kit comprises: (a) at least one agent for
detecting a biomarker
of a nucleic acid molecule having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or
99% SEQ ID NO. 7 or SEQ ID NO. 8 and (b) instructions to detect the marker or
markers by
contacting a sample with the adsorbent and detecting the marker or markers
with a biological
sample. In some embodiments, the kit may comprise an eluant (as an alternative
or in
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combination with instructions) or instructions for making an eluant, wherein
the combination of
the adsorbent and the eluant allows detection of the markers using gas phase
ion spectrometry.
Such kits can be prepared from the materials described above, and the previous
discussion of
these materials (e.g., probe substrates, adsorbents, washing solutions, etc.)
is fully applicable to
5 this section and will not be repeated.
[0046] In one embodiment, the kit comprises a first substrate
comprising an adsorbent
thereon (e.g., a particle functionalized with an adsorbent) and a second
substrate onto which the
first substrate can be positioned to form a probe which is removably
insertable into a gas phase
ion spectrometer. Alternatively, the kit may comprise a single substrate which
is in the form of a
10 removably insertable probe with adsorbents on the substrate. The kit may
further comprise a
pre-fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA
agarose column,
size exclusion column, Q-anion exchange spin column, single stranded DNA
column, lectin
column, etc.).
[0047] Optionally, the kit can further comprise instructions for
suitable operational
15 parameters in the form of a label or a separate insert. For example, the
kit may have standard
instructions informing a consumer how to wash the probe after a sample is
contacted on the
probe. In another example, the kit may have instructions for pre-fractionating
a sample to reduce
complexity of proteins in the sample. In another example, the kit may have
instructions for
automating the fractionation or other processes.
Biological Fluids
[0048] The inventive compositions of matter, processes, methods and in
vitro diagnostic
devices provide the ability to detect and monitor levels of those proteins or
autoantibodies which
are released into the body after injury to provide enhanced diagnostic
capability by allowing
clinicians (1) to determine the level of injury severity in patients with
various injuries, (2) to
monitor patients for development of disorders or signs of secondary injuries
that may elicit these
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cellular changes and (3) to continually monitor the progress of the injury and
the effects of
therapy by examination of these proteins in biological fluids, such as blood,
plasma, serum, CSF,
urine, saliva or sweat.
[0049] A sample is preferably a biological sample. Preferred examples
of biological
samples are illustratively cells, tissues, cerebral spinal fluid (CSF),
artificial CSF, whole blood,
serum, plasma, cytosolic fluid, urine, feces, stomach fluids, digestive
fluids, saliva, nasal or other
airway fluid, vaginal fluids, semen, buffered saline, saline, water, or other
biological fluid
recognized in the art. Preferably, the biological samples comprise CSF, blood,
serum, plasma,
sweat, saliva and urine. It should be appreciated that after injury, the cell
membrane in any
muscle, tissue or organ becomes compromised, leading to the efflux of its
constituent proteins
first into the extracellular fluid or space and eventually into other fluids
in the body, including
the circulating blood. Through normal bodily function (such as impurity
removal from the
kidneys), the proteins migrate to other biological fluids such as urine,
sweat, and saliva. Thus,
other suitable biological samples include, but not limited to such cells or
fluid secreted from
these cells. It should also be appreciated that obtaining biological fluids
such as cerebrospinal
fluid, blood, plasma, serum, saliva and urine, from a subject is typically
much less invasive and
traumatizing than obtaining a solid tissue biopsy sample. Thus, samples, which
are biological
fluids, are preferred for use in the invention.
[0050] Biological samples of CSF, blood, urine and saliva are collected
using normal
collection techniques. For example, and not to limit the sample collection to
the procedures
contained herein, CSF Lumbar Puncture (LP) a 20-gauge introducer needle is
inserted and an
amount of CSF is withdrawn. For blood, the samples may be collected by
venipuncture in
Vacutainer tubes, and if preferred spun down and separated into serum and
plasma. For urine
and saliva, samples are collected avoiding the introduction of contaminants
into the specimen is
preferred. All biological samples may be stored in aliquots at -80 C for later
assay. Surgical
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techniques for obtaining solid tissue samples are well known in the art. Any
suitable biological
samples can be obtained from a subject to detect markers. It should be
appreciated that the
methods employed herein may be identically reproduced for any biological fluid
to detect a
marker or markers in a sample.
EXAMPLES
[0051] Various aspects of the present invention are illustrated by the
following non-limiting
examples. The examples are for illustrative purposes and are not a limitation
on any practice of
the present invention. It will be understood that variations and modifications
can be made
without departing from the spirit and scope of the invention. While the
examples are generally
directed to mammalian tissue, specifically, analyses of mouse tissue, a person
having ordinary
skill in the art recognizes that similar techniques and other techniques known
in the art readily
translate the examples to other mammals such as humans. Reagents illustrated
herein are
commonly cross reactive between mammalian species or alternative reagents with
similar
properties are commercially available, and a person of ordinary skill in the
art readily
understands where such reagents may be obtained. Variations within the
concepts of the
invention are apparent to those skilled in the art.
[0052] EXAMPLE 1
[0053] Three clusters were tested for increasing size of mRNAs (mRNAs
were converted to
cDNAs to prevent enzymatic deterioration of the RNA sequences). Cluster 1
focused on testing
for and sequencing all mRNAs that were from about 1,000 to 2,000 bases in
length, give or take
several hundred bases (see histograms). Clusters 2 and 3 were from 2,000 to
3,000 bases and
3,000 to 6,000 bases, respectively.
[0054] The report for cluster 1 contained 9,137 pages of sequences like
the one below
representing about 25,000 to 30,000 potentially unique mRNA sequences and thus
proteins. The
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one below is the mRNA sequence for S10007 (the 13714th sequence tested and
sequenced in
cluster 1).
>c13714/f2p4/850
isoform=c13714;full length coyerage=2;non full length coyerage=4;isoform
length=850
cCGCCCAGGACCCGCAGCAGAGACGACGCCTGCAGCAAGGAGACCAGGAAGGGGTGAGAC
AAGGAAGAGGATGTCTGAGCTGGAGAAGGCCATGGTGGCCCTCATCGACGTTTTCCACCA
AT AT IdT GGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCGAACT CAAGGAGC T CAT
CAACAAT GAGCTT TCCCAT TT CT TAGAGAGATGGAGT CT CCCTATGTT GCCCAGGCTGGT
CTCAAACTTCCTGTGCTCAAGTTATCCTCCTGCCTTAGCTTCCCAAAGTGCTGGGATTAC
AGGAAAT CAAAGAGCAGGAGGTT GT GGACAAAGT CAT GGAAACACT GGACAATGAT GGAG
ACGGCGAATGTGACTTCCAGGAATTCATGGCCTTTGTTGCCATGGTTACTACTGCCTGCC
ACGAGTTCTTTGAACATGAGTGAGATTAGAAAGCAGCCAAACCTTTCCTGTAACAGAGAC
GGTCATGCAAGAAAGCAGACAGCAAGGGCTTGCAGCCTAGTAGGAGCTGAGCTTTCCAGC
CGTGTTGTAGCTAAT TAGGAAGCTT GATT TGCTT T GT GATTGAAAAAT TGAAAACCTCTT
TCCAAAGGCTGTTTTAACGGCCTGCATCATTCTTTCTGCTATATTAGGCCTGTGTGTAAG
CT GACTGGCCCCAGGGACT CT TGTTAACAGTAACT TAGGAGT CAGGTCTCAGTGATAAAG
CGTGCACCGTGCAGCCCGCCATGGCCGTGTAGACCCTAACCCGGAGGGAACCCTGACTAC
AGAAATTACCCCGGGGCACCCTTAAAACT TCCACTACCT TTAAAAAACAAAGCCTTAT CC
AGCATTATTT
EXAMPLE 2
[0055] ELISA assays were performed with sera from eight humans, two
with no known
history of neural cell damage, and six presenting with one or more clinical
symptoms of an
injury or disease resulting from neural cell damage (three with traumatic
brain injury, one with
stroke, one with Alzheimer's Disease). Microplates were coated with antibodies
raised against
the sequence of SEQ ID NO 7. Wells were washed. One hundred !.1.1 serum from
the test subjects
was added to each well. The micropiates were incubated at 37 C. for one hour
and then washed
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again to remove any unbounded antibodies.
The protein-antibody complex was then
subsequently measured. In comparing the levels of the protein-antibody complex
formed by the
:ELISA method, it was found that the six subjects presenting with one or more
clinical symptoms
of a disease or injury resulting from neural cell damage had significantly
higher concentrations
of the protein-antibody matrix formed in the ELISA method than the two
uninjured patients.
EXAMPLE 3
00561
The test of Example 2 was repeated with antibodies raised against the
sequence of
SEQ ID NO 8. ELBA. Similarly, in comparing the levels of the protein-antibody
complex
formed by the ELBA method, it was found that the six subjects presenting with
one or more
clinical symptoms of a disease or injury resulting from neural cell damage had
significantly
higher concentrations of the protein-antibody matrix formed in the EL-ISA
method than the two
uninjured patients.
Other Embodiments
[0057]
While at least one exemplary embodiment has been presented in the foregoing
detailed description, it should be appreciated that a vast number of
variations exist. It should also
be appreciated that the exemplary embodiment or exemplary embodiments are only
examples,
and are not intended to limit the scope, applicability, or configuration of
the described
embodiments in any way. Rather, the foregoing detailed description will
provide those skilled in
the art with a convenient road map for implementing the exemplary embodiment
or exemplary
embodiments. It should be understood that various changes can be made in the
function and
arrangement of elements without departing from the scope as set forth in the
appended claims
and the legal equivalents thereof
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[0058] Patent documents and publications mentioned in the specification
are indicative of
the levels of those skilled in the art to which the invention pertains. These
documents and
publications are incorporated herein by reference to the same extent as if
each individual
document or publication was specifically and individually incorporated herein
by reference.
5 [0059] The foregoing description is illustrative of particular
embodiments of the invention
but is not meant to be a limitation upon the practice thereof The following
claims, including all
equivalents thereof, are intended to define the scope of the invention.
