Note: Descriptions are shown in the official language in which they were submitted.
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DIFFERENTIAL IMMUNOASSAY
BACKGROUND OF THE INVENTION
Innumerable qualitative and quantitative tests are available for detecting the
presence or level
of particular substances in a sample. Sources of such samples range from
industrial
enviromnents such as mines, wastewater processing, food quality, soil testing,
among many
others. In the medical field, tests for substances in bodily fluids are well
known, and are aids
to prognostication, diagnosis, and monitoring the progression and treatment of
various
conditions and diseases. In many cases, multiple tests are performed on a
sample and a
health care professional then makes a presumptive diagnosis based on the
various levels of
particular analytes in the sample, among other information gained, for
example, from
examining a patient.
In certain circumstances, particular in the emergency room and ambulance call,
time is of the
essence in arriving at a diagnosis and initiating appropriate therapy to
intervene in the
morbidity and mortality of a rapidly deteriorating condition. One such example
is diagnosis
of a heart attack in an individual with chest pain or recent onset. Multiple
diagnoses may be
attributable to chest pain, yet diagnosis based on electrocardiogram or levels
of cardiac
markers released into the circulation are needed for a confirmatory diagnosis
and initiation of
a course of therapy, which would be unwise in a patient not having a heart
attack. Thus, the
need for rapid and accurate, early diagnostic tests is apparent for such
emergency conditions.
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Although such early tests are available, even such tests are not without
flaws. For example,
diagnosis of a heat attack within six hours of the onset of chest pain is
difficult to perform
with a single test. While the cardiac marlcer troponin I has been recently
adopted as a single
and highly accurate indicator, it is not detectable until after about six
hours, leaving a large
window where early initiation of treatment would be highly desirable but
dangerous without
an accurate diagnosis. Another cardiac marker, myoglobin, is released into the
circulation
earlier than troponin I, but is not specific for cardiac tissue, as skeletal
muscle damage also
releases myoglobin into the circulation. Additional tests may be performed
together with
myoglobin to attempt to identify its origin, in order to improve the accuracy
of an early
to diagnosis.
The foregoing example of heart attack is merely one example of a myriad of
diagnoses,
which if to be carned out with a high degree of accuracy, need additional,
corroborative tests.
Although the combination of multiple assays performed simultaneously increases
diagnostic
15 precision, it is undesirable in that it also increases the complexity of
the testing, the
coordination of the timing of the separate test procedures and availability of
the information,
and the amount of information that must be processed manually or otherwise,
often under
emergency conditions.
20 It is towards the simplification of multiple analyte diagnostic tests to
provide a single readout
reliably indicative of a particular diagnosis that the present invention is
directed.
BRIEF DESCRIPTION OF THE 1NVENTION
In its broadest aspect, the present invention provides an assay useful to
determine the relative
25 levels at which different analytes are present in a sample. In the present
assay, the extent of
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the readout is related to the level of the first analyte and that of the
second analyte. Thus, in
one embodiment, the readout provides either a ratio of the level of the first
analyte to the
second, or the difference in levels between the first analyte and the second
analyte.
The assay of the invention is useful for situations in which a ratio or
difference between the
levels of the first and second analytes is diagnostically useful, and a single
readout that takes
the two values into consideration in generating a single differential value
can be as
informative and directive of further action as would be obtaining the
individual values and
mentally evaluating or arithmetically calculating the difference or ratio, and
then acting upon
to the result. The method of the invention simplifies decision making by
internally integrating
the results of at least two individual analyte levels.
By way of non-limiting example, the first analyte and second analytes may be
markers useful
for determining the health status of an individual, wherein the ratio or
difference among the
is markers is diagnostically informative. In a particular embodiment, elevated
levels of the first
analyte may be indicative of a life-threatening medical event only if the
level of the second
analyte is not elevated. In another embodiment, the second analyte also being
elevated is
indicative of an event. In a third and preferred embodiment, an analyte may
originate from
different bodily sources and the origin is diagnostically useful; the assay of
the invention is
2o useful for identifying the source of elevated levels of the first analyte.
In this embodiment,
the format of the assays of the invention takes advantage of the co-release of
another analyte
from the bodily source other than the intended source (referred to herein as a
non-target-
source marl~er) whose level is effectively subtracted from that of the total
level of desired
analyte to provide in a single test, a readout specific to the analyte source.
2s
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In one broad aspect, the single assay for a preselected analyte is indicated
as the level of the
first analyte reduced proportionally by the level of a second analyte present
in the sample. A
reading is obtained only if the first analyte is present, and the detected
level of the first
analyte is reduced as the level of the second analyte increases.
In the present assay, the relative presence or level of first and second
analytes in a given
sample is revealed by utilizing a labeling reagent for one of the two analytes
that labels that
analyte through a reaction that is inhibited by the other analyte. More
particularly, in the
present assay, the readout is dependent on binding, to one of the analytes
selectively, of a
to labeling reagent complex the formation of which is inhibited by any second
analyte present in
the sample. Thus, labeling of the analyte targeted by the labeling reagent
proceeds in the
absence of the second analyte, and a reading is obtained. No reading is
obtained when the
first analyte is absent from the sample. When both analytes are present in the
sample, the
labeling reaction proceeds but in a manner that is competitively inhibited by
the second
15 analyte. Thus, the relative levels at which the first and second analytes
are present in the
sample is reflected by the extent to which the firs~analyte is labeled, and
this is ultimately
reflected in the readout obtained following performance of the assay.
Thus, in one of its aspects, the present invention provides a method useful to
assay a sample
2o to detect the presence or relative levels therein of first and second
analytes, the method
comprising the step of bringing the sample into contact with a labeling
reagent means adapted
to form a labeling complex that binds to and thereby labels the first analyte,
wherein
formation of the labeling complex is inhibited by second analyte present in
the sample.
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In a preferred aspect of the invention, the labeling reagent means comprises
two components:
a labeled binding partner for the second analyte, and a conjugate formed by
coupling of a
second analyte itself and a binding partner for the first analyte. When second
analyte is
absent, the first analyte is thus labeled by formation of complexes between
the first analyte
and the first analyte binding partner, and the second analyte and the labeled
second analyte
binding partner. When present in the sample, however, the second analyte
becomes a
competitor for binding to the labeled second analyte binding partner, and
thereby inhibits
binding of that labeled second analyte binding partner to the conjugate, thus
reducing labeling
of the first analyte.
In a particular embodiment, a method is provided for identifying in a sample
the presence or
level of a first analyte above the level of a second analyte comprising the
steps of
(a) forming a reaction mixture by contacting the sample with reagent means for
labeling the first analyte, the labeling reagent means comprising a mobile,
is labeled binding partner to the second analyte, and a conjugate between the
second analyte and a binding partner to the first analyte;
(b) contacting the reaction mixture with an immobilized binding partner to the
first analyte;
wherein the extent of formation of a complex comprising the mobile, labeled
binding partner
2o to the second analyte, the conjugate between the second analyte and the
binding partner to the
first analyte, the first analyte, and the immobilized binding partner to the
first analyte, is
indicative of the presence or level of the first analyte in the sample reduced
by the level of the
second analyte in the sample. Desirably, the reaction is staged by first
bringing the sample
into contact with the mobile, labeled binding partner to the second analyte to
allow any
25 second analyte in the specimen to become bound thereto, and then presenting
the conjugate
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before finally contacting the resultant mixture with immobilized binding
partner to the first
analyte. In this way, the inhibitory effect of sample-borne second analyte is
maximized, by
allow it to react first with the labeled second analyte binding partner before
allowing the
conjugate to compete therewith for binding.
By way of non-limiting example, the aforementioned binding partners may be
antibodies.
The label may be colloidal gold. The sample may be, by way of non-limiting
example, a
bodily fluid, wastewater, a foodstuff; preferably, it is a bodily fluid such
as whole blood,
serum, plasma, or urine. By way of example, the first analyte may be a cardiac
marker, such
l0 as myoglobin, and the second analyte may be a different analyte co-released
from a non-
cardiac source along with the first analyte, such as carbonic anhydrase III
which is released
from damaged skeletal muscle along with myoglobin. For determining the level
of
myoglobin originating from the heart, the mobile, labeled binding partner to
the second
analyte may be a gold-labeled monoclonal anti-carbolic anhydrase III antibody,
the
15 conjugate between the second analyte and a binding partner to the first
analyte may be a
conjugate between carbonic anhydrase III and an anti-myoglobin monoclonal
antibody, and
the immobilized binding partner to the first analyte may be an anti- myoglobin
monoclonal
antibody.
2o The conjugate between the second analyte and a binding partner to the first
analyte may be a
covalent conjugate between the members, such as is achievable using a
homobifunctional or
heterobifunctional cross-linking agent or carbodiimide, or it may comprise a
single-chain
polypeptide on which reside both the second analyte, or an epitope thereof,
and a binding
partner, or binding portion thereof, to the first analyte, such that each
member retains its
25 desired activities within the conjugate or single-chain polypeptide. For
example, the
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conjugate between an antibody to myoglobin and carbonic anhydrase III may
include a
single-chain polypeptide comprising carbonic anhydrase III and the
immmloglobulin heavy
chain, which when assembled into the functioning antibody, provides binding
sites for
myoglobin and a carbonic anhydrase III portion to which the labeled anti-
carbonic anhydrase
III antibody may bind. The analyte portion of any of the conjugates herein may
be the full-
length analyte or a fragment bearing the epitope recognized by the
corresponding binding
partner. The foregoing example may be used to diagnose a heart attack by
indicating an
elevated level of myoglobin exists over that which may derived from a non-
cardiac source.
In this case, the level of cardiac and skeletal (i.e., total) myoglobin
detected in the assay is
reduced by the amount of carbonic anhydrase III present in the sample, the
latter equivalent
to the level of skeletal muscle-derived myoglobin.
In a second embodiment, a homogeneous assay similar to that above is provided
which
employs slightly different reagents, but applies the same principles. W this
embodiment, a
further binding interface is incorporated into the labeling reaction.
Particularly, the conjugate
between the first analyte binding partner and the second analyte instead
introduces a further
biotin/streptavidin interaction, and the conjugate thus is represented by two
reagents; one in
which biotin is conjugated with either the first analyte antibody or the
second analyte, and
another in which streptavidin or a biotin-binding component thereof is
conjugated with the
other of the first analyte antibody or the second analyte. In an embodiment,
the conjugate
reagents are a first conjugate between first analyte antibody and biotin, and
a second
conjugate between streptavidin and the second analyte.
Thus, a method is provided for identifying in a sample the presence or level
of a first analyte
above a second analyte, the method comprising conducting an assay following
the steps of
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(a) forming a reaction mixture by contacting the sample with
(1) a mobile, labeled binding partner to the second analyte,
(2) a conjugate between the second analyte and streptavidin; and
(3) a biotinylated binding partner to the first analyte; and then
(b) contacting the reaction mixture with an immobilized binding partner to the
first analyte;
wherein the extent of formation of a complex comprising the mobile, labeled
binding partner
to the second analyte, the conjugate between the second analyte and
streptavidin, the
biotinylated binding partner to the first analyte, the analyte, and the
immobilized binding
to partner to the first analyte, is indicative of the presence or level of the
first analyte in the
sample reduced by the level of the second analyte in the sample.
Desirably, the assay is performed by staging the addition of reagents in step
(a), so that
sample is first exposed to the mobile labeled binding partner to the second
analyte so that any
15 second analyte in the sample becomes bound thereto, before addition of the
competitive-
binding conjugate between streptavidin and the second analyte.
By way of non-limiting example, the aforementioned binding partners may be
antibodies.
The label may be colloidal gold. Streptavidin, or a biotin-binding component
thereof, or
2o another biotin-binding partner may be used. The sample may be a biological
sample such as
a bodily fluid: examples include whole blood, serum, plasma, and urine. By way
of example,
the first analyte may be a cardiac marker, such as myoglobin, and the second
analyte may be
a different analyte co-released from a non-cardiac source along with the first
analyte, such as
carbonic anhydrase III released from damaged skeletal muscle along with
myoglobin. For
25 determining the level of myoglobin originating from the heart, the mobile,
labeled binding
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partner to the second analyte may be a gold-labeled monoclonal anti-carbonic
anhydrase III
antibody, the conjugate between the second analyte and a biotin-binding
molecule a
conjugate between carbonic anhydrase III and streptavidin, a conjugate between
biotin and a
binding partner to the first analyte may be a biotinylated anti-myoglobin
monoclonal
antibody, and the immobilized binding partner to the first analyte may be an
anti-myoglobin
monoclonal antibody.
The conjugate between the second analyte and a binding partner to biotin may
be a covalent
conjugate between the members, such as is achievable using a homobifunctional
or
to heterobifimctional cross-linking agent or carbodiimide, or it may comprise
a single-chain
polypeptide on which reside both the second analyte or an epitope thereof, and
streptavidin or
the biotin-binding portion thereof, such that each member of the conjugate of
single-chain
polypeptide independently retains its respective binding activity. For
example, the conjugate
between streptavidin and carbonic anhydrase III may be a single-chain
polypeptide
15 comprising carbonic anhydrase III, or an epitope thereof, and streptavidin
or a biotin-binding
portion thereof, thus providing binding sites for both a biotinylated antibody
and anti-
carbonic anhydrase III antibody. As mentioned above, the analyte portion of
any of the
conjugates herein may be the full-length analyte or a fragment bearing the
epitope recognized
by the binding partner. The foregoing example may be used to diagnose a heart
attack by
2o indicating an elevated level of myoglobin exists over that which may
derived from a non-
cardiac source, in the same manner as described in the previous embodiment.
Variations on
these embodiments in the selection of the reagents and operation of the
components of the
test are fully embraced within the spirit and scope of the present invention.
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In a preferred embodiment, the first or preselected analyte is an analyte
originating from a
target source whose level is desirably measured over the same analyte
originating from a
source other than the target source. A second analyte is a marlcer that is
released from the
non-target (other) source in proportion to the level of first analyte released
from the non-
target source. The assay of the invention subtracts or proportionally reduces,
depending on
binding partner affinities, from the total level of first analyte (from the
target and non-target
source) the level of the second analyte, which effectively subtracts the level
of the first
analyte derived from the non-target source from the readout value.
to By selecting the affinities of the binding partners to the analytes and
ratio of the components
in the conjugates of the invention, as well as using fragments of the analytes
comprising the
epitope of the analyte recognized by the binding partners, the relative
sensitivity of the assay
to the first analyte and especially the reduction in value achieved by the
presence of any
second analyte in the sample may be adjusted to provide an assay which
essentially reads out
15 the ratio between the first analyte and the second analyte. For example, if
the second analyte
is released from the non-target tissue in very small amounts compared to the
amount of the
target analyte released from the non-target source, use of a higher affinity
antibody in the
conjugate of the invention to the second analyte in contrast to a lower
affinity antibody to the
first analyte will increase the sensitivity of the assay to any second analyte
present in the
2o sample. Such variations in the invention are within the realm embraced
here, and one of
skill in the art by following the teachings herein will readily prepare an
assay for other
analytes or with other operating characteristics, sensitivities, ranges, or
other parameters.
Thus, in one embodiment, a method is provided for identifying in a sample the
presence or
25 level of a preselected analyte originating from a target source, wherein
any level of the
to
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preselected analyte in the sample originating from a source other than the
target source is
associated with an increased level in the sample of a marker from the source
other than the
target source, the method comprising conducting an assay following the steps
of
(a) contacting the sample with a labeling reagent comprising (1) a mobile,
labeled
binding partner to one of the preselected analyte and the marker, (2) a
conjugate between the marker and a binding partner to the preselected analyte;
and then
(c) contacting the resulting sample with an immobilized binding partner to the
other of the marker and the preselected analyte;
1 o wherein the extent of labeling of said immobilized binding partner is
indicative of the
presence or level of the preselected analyte in the sample reduced by the
level of the marker
originating from the source other than the target source.
In a preferred embodiment, the immobilized binding partner is a binding
partner for the
15 analyte, and the labeled, mobile binding partner is a binding partner for
the marker.
By way of non-limiting example, the aforementioned binding parhiers may be
antibodies.
The label may be colloidal gold. The sample may be a biological sample such as
whole
blood, serum, plasma, or urine. By way of example, the preselected analyte may
be a cardiac
20 analyte, such as myoglobin, and the corresponding marker may be carbonic
anhydrase III.
For determining the level of myoglobin originating from the heart, the mobile,
labeled
binding parhler to said marker may be a gold-labeled monoclonal anti-carbonic
anhydrase III
antibody, the conjugate between the marker and a binding partner to the
preselected analyte
may be a conjugate between carbonic anhydrase III and an anti-myoglobin
monoclonal
25 antibody, and the immobilized binding partner to the preselected analyte
may be a~z anti-
11
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myoglobin monoclonal antibody. The conjugate between the marker and a binding
partner to
the preselected analyte may be a covalent conjugate between the members, such
as is
achievable using a homobifunctional or heterobifunctional cross-linking agent
or
carbodiimide, or it may comprise a single-chain polypeptide on which reside
both the marker
or an epitope thereof and a binding partner or portion thereof, such that each
member retains
the desired activities in the conjugate or single-chain polypeptide. For
example, the
conjugate between an antibody to myoglobin and carbonic anhydrase III may
include a
single-chain polypeptide comprising carbonic anhydrase III and the
immunoglobulin heavy
chain, which when assembled into the functioning antibody, provides binding
sites for
to myoglobin and a carbonic anhydrase III portion to which the labeled anti-
carbonic anhydrase
III antibody may bind. The foregoing example may be used to diagnose a heart
attack, as
described above.
In a second embodiment, an assay similar to that above is provided which
employs variations
15 in the components, but provides the same objectives. Thus, a method is
provided for
identifying in a sample the presence or level of a preselected analyte
originating from a target
source, wherein any level of said preselected analyte in the sample
originating from a source
other than the target source is associated with an level in the sample of a
marker from the
source other than the target source, the method comprising conducting an assay
following the
2o sequential steps of
(a) first contacting the sample with an analyte labeling reagent comprising
(1) a mobile, labeled binding partner to one of the preselected analyte or
the marker,
(2) a conjugate between the marker and one of biotin and streptavidin, and
12
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(3) a binding partner to the preselected analyte conjugated to the other of
biotin and streptavidin; and then
(b) contacting the sample with an immobilized binding partner to the other of
the
preselected analyte and the marlcer;
wherein the extent of labeling of the immobilized binding partner is
indicative of the presence
or level of the preselected analyte in the sample reduced by the level of the
marker in the
sample originating from the source other than the target source .
In a preferred embodiment, the analyte labeling reagent comprises (1) a
mobile, labeled
to binding partner to the marker, (2) a conjugate between the marker and
streptavidin, and (3) a
biotinylated binding partner to the preselected analyte.
In other preferred embodiments, the immobilized binding partner is a binding
partner for the
analyte.
By way of non-limiting example, the aforementioned binding partners may be
antibodies.
The label may be colloidal gold. The sample may be a biological sample such as
whole
blood, serum, plasma, or urine. By way of example, the preselected analyte may
be a cardiac
analyte, such as myoglobin, and the corresponding marker may be carbonic
anhydrase III.
For determining the level of myoglobin originating from the heart, the mobile,
labeled
binding partner to the marker may be a gold-labeled monoclonal anti-carbonic
anhydrase III
antibody, the conjugate between the marker and streptavidin may be a conjugate
between
carbonic anhydrase III and streptavidin, the biotinylated binding partner to
the preselected
analyte may be biotinylated anti-myoglobin monoclonal antibody, and the
immobilized
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binding partner to the preselected analyte may be an immobilized anti-
myoglobin monoclonal
antibody.
The aforementioned conjugate between the marker and streptavidin may be a
covalent
conjugate prepared, for example, by use of a homobifunctional or
heterobifunctional cross-
linl~ing agent or carbodiimide, or may be a single-chain polypeptide on which
reside both the
marker or an epitope thereof and streptavidin, each retaining its desired
activities and ability
to participate in the above-mentioned assay. The foregoing example may be used
to diagnose
a heart attack.
to
Of course, in the above methods, wherein two binding partners bind to the
preselected
analyte, each must be capable of recognizing a different binding site on the
preselected
analyte such that both binding partmers can independently bind and permit the
final labeled
complex to form if the second analyte (marker) is not present at a level
relatively greater than
15 that of the first analyte. Moreover, the sensitivities and selectivities of
the foregoing assays
may be adjusted, for example, depending on the relative levels of the
preselected analyte
released from the target source, the level released from the non-target
source, and the amount
of co-release of the non-target-source marker relative to the release of the
preselected analyte
from the non-target source. The ratios of the components in the various
reagents of the
2o assays may be adjusted, and any reduced binding thereby compensated for in
another reagent,
as an example of the flexibility of the assay for various analytes.
Preferably, the foregoing binding partners are antibodies, and may be
monoclonal or
polyclonal antibodies. The preselected analyte is preferably a biomolecule,
such as a protein,
25 carbohydrate, nucleic acid, lipid, glycoprotein, glycolipid, by way of
example, but it is not so
14
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limited. The preselected analyte is capable of being recognized by the binding
of two
different binding partners, preferably antibodies. The preselected analyte may
be present in
any sample, including that from a human or animal body, foodstuff or food
processing or
manufacturing facility, domestic or industrial water supply, etc. Preferably,
the sample is a
bodily fluid from a hmnan. In a preferred embodiment, the sample is whole
blood, the
preselected analyte is myoglobin, and the marker is carbonic anhydrase III.
The marker that is also present in the sample is preferably a biomolecule,
such as a protein,
carbohydrate, nucleic acid, lipid, glycoprotein, glycolipid, by way of
example, but it is not so
to limited. The marker is capable of binding to a binding partner for the
marker, preferably an
antibody, and the presence of any marker in the sample is capable of competing
for binding
to the binding partner to the marker with a conjugate comprising the marker,
as described
above.
15 The multiple steps in the foregoing examples of the assay of the invention
described above
may be best illustrated by specific example. Elevated levels of circulating
myoglobin, a
cardiac and skeletal muscle protein, may be diagnostic for a heart attack if
the myoglobin is
of cardiac (heart) origin and not from skeletal muscle. Elevated circulating
myoglobin from
skeletal muscle may indicate muscle damage. While other, more specific cardiac
markers are
2o available, myoglobin is particularly desirable if its source can be
determined, as it is released
early following heart muscle damage, in contrast to other more specific
cardiac marlcers,
which are detectable later (for example, after six hours). The method of the
present invention
provides the level of myoglobin of cardiac origin over that of skeletal origin
by taking
advantage of the simultaneous release from skeletal muscle of carbonic
anhydrase III (CAIII)
25 together with myoglobin. Thus, the first method described hereinabove
employs a test strip
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with 1) a mobile, labeled anti-carbonic anhydrase III antibody; 2) a mobile
covalent or single-
chain polypeptide-containing conjugate between carbonic anhydrase III and anti-
myoglobin
antibody; and 3) anti-myoglobin antibody immobilized at the capture zone. In
the presence
of myoglobin in the sample, myoglobin will form an immunocomplex with the
mobile
carbonic anhydrase III-anti-myoglobin antibody conjugate, which will be
captured at the
capture zone by the immobilized anti-myoglobin antibody. The mobile, labeled
anti-carbolic
anhydrase III antibody will bind to the carbonic anhydrase III on the
conjugate, forming a
positive band due to the presence of the label. However, in the presence of
both myoglobin
and carbonic anhydrase III in the sample, the level of binding of the labeled
anti-carbonic
to anhydrase antibody to the mobile carbonic anhydrase III-anti-myoglobin
antibody conjugate
will be reduced and therefore less will be available to bind to the carbonic
anhydrase III on
the conjugate, and a reduced or negative result will be obtained, depending on
the amount of
carbonic anhydrase III present in the sample.
is In the second method described above which employs slightly different
reagents, myoglobin
in the sample will form an immunocomplex with the biotinylated anti-myoglobin
antibody,
which will then be captured at the capture line by the immobilized anti-
myoglobin antibody.
The conjugate (covalent or single-chain polypeptide) between streptavidin and
carbonic
anhydrase III will bind to the biotinylated antibody at the capture line, and
the mobile,
2o labeled anti-carbonic anhydrase III will further bind to the streptavidin-
carbonic anhydrase III
conjugate in the complex, forming a positive band. However, in the presence of
both
myoglobin and carbonic anhydrase III, the level of binding of the mobile,
labeled anti-
caxbonic anhydrase III antibody to the streptavidin-carbonic anhydrase III
conjugate will be
reduced by the any carbonic anhydrase III in the sample, and therefore will no
longer be
16
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available to bind to the immobilized complex. A reduced or negative result
will be obtained,
depending on the amount of carbonic anhydrase III in the sample.
Depending on antibody affinities, sample flow, volumes, and other parameters,
the foregoing
assay reduces myoglobin detectability approximately to the extent of the
presence of carbonic
anhydrase III, in a reciprocal relationship. Modifications of the foregoing
methods which
achieve the same objectives are lilcewise embraced herein.
It will further be appreciated that formats alternative to the lateral flow,
strip-based format
1o can also be utilized to perform the present assay. In particular, and in
embodiments of the
invention, the assay is performed in ELISA format, using for instance standard
microwell
trays conveiuent for use in robotic readers. In this format, for instance, the
sample is mixed
with the analyte labeling reagent, and the mixture is then contacted with a
binding partner for
one of the analytes, desirably the preselected analyte that has been
immobilized in the
15 standard way. Following incubation to allow formation of the labeling
complex, unbound
sample is removed by washing, and a reading is then taken of the immobilized
label.
Other pairs of first and second analytes for which knowledge of a ratio or
difference in levels
is diagnostically useful include fatty acid binding protein and carbonic
anhydrase III, and
2o myosin light chain and carbonic anhydrase III for the diagnosis of heart
attack; and total
cholesterol and high-density lipoprotein (HDL) for assessing risk of
atherosclerotic diseases.
The invention is also directed to conjugates or single-chain polypeptides
comprising a biotin-
binding protein or protein fragment, such as streptavidin, and an analyte, or
anti-analyte-
25 binding epitope thereof, to provide a reagents useful in the practice of
the invention. For
17
CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
example, a single-chain polypeptide comprising streptavidin and carbonic
anhydrase III is
useful as the reagent which can bind both a biotinylated antibody and an
antibody to carbonic
anhydrase III, the utility of which in the practice of the invention is
evident from the
teachings herein. An example is the single-chain polypeptide shown in SEQ ID
NO:1, but
this is merely illustrative of a wide variety of conjugates of analytes and
biotin-binding
molecules that are embraced herein, and may be further extended beyond biotin-
streptavidin
to other high-affinity binding pairs between one molecule and another, each of
which may be
separately incorporated into reagents and retain their binding activity. The
foregoing
conjugates preferably may be prepared by recombinant techniques, wherein a
single-chain
to polypeptide comprising the analyte and streptavidin, joined by a linker
peptide, are expressed
from a single polynucleotide construct. The invention further embraces
polynucleotide
sequences encoding such conjugates, such as those that encode SEQ ID NO:1.
Alternatively,
cross-linking agents may be used to form the reagent. Such include
homobifunctional,
heterobifunctional, carbodiimides, and such conjugation methods involving
covalently
15 liu~ing, with or without a spacer, one functional group of a biomolecule to
another is well
known in the art.
Another reagent embraced by the present invention is a conjugate of single-
chain polypeptide
comprising a portion of an antibody (or antigen-binding domain thereof) and a
biotin-binding
2o protein or fragment thereof, such that the conjugate, single-chain
polypeptide or full antibody
may independently recognize and bind to both its epitope and to biotin. The
use of this
reagent in the practice of the present invention will be evident from the
teachings herein. By
was of non-limiting example, a reagent comprising streptavidin or a biotin-
binding portion
thereof covalently bound to an anti-carbonic anhydrase antibody, is described.
These
25 reagents may be made by, for example, a bifunctional cross-linking reagent
to covalently
18
CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
bind the members of the reagent together, or it may be prepared by recombinant
methods, for
example, in the construction of a polynucleotide that expresses an
immwoglobulin heavy
chain with a biotin-binding fragment of streptavidin fused, with our without a
linker
sequence, to the C-terminal portion of the heavy chain. Association of this
single-chain
hybrid immunoglobulin molecule with the immunoglobulin light chain will
provide a
modified antibody molecule capable of both recognizing and binding the
intended epitope,
and also binding to biotin. This is merely illustrative of methods of
preparation and is not
intended in any way to be limiting.
to The invention also embraces polynucleotide sequences encoding such single-
chain
polypeptide compositions comprising an immunoglobulin light or heavy chain and
a biotin-
binding protein or peptide such as streptavidin. A non-limiting example is a
polynucleotide
sequence which encodes SEQ ID NO:1.
15 Thus, the invention is also directed to a conjugate comprising an analyte
or a fragment
thereof, and streptavidin or a biotin-binding equivalent thereof e.g., a
biotin-binding variant
or fragment of streptavidin, wherein independently, the analyte or fragment
thereof in the
conjugate is capable of being bound by an antibody to the analyte, and said
streptavidin or
biotin-binding fragment thereof in the conjugate is capable of binding to
biotin. In a
2o preferred embodiment, the analyte or fragment thereof is a protein or
peptide. hl a further
embodiment, the protein or peptide analyte or fragment thereof and
streptavidin or a biotin-
binding fragment thereof reside on a single polypeptide chain. In a non-
limiting example, the
analyte is carbonic anhydrase III. An example of such a single-chain
polypeptide is depicted
in SEQ ID NO:1.
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WO 02/16943 PCT/IBO1/01531
In another embodiment, the invention is directed to conjugate comprising (1)
an antibody to a
first analyte or a binding fragment thereof, and (2) a second analyte (marker)
or a fragment or
variant thereof which competes with the second analyte for binding to antibody
to the second
analyte. In conjugated form, the antibody component in the conjugate is
capable of binding
the first analyte, and the second analyte component is capable of being bound
by an antibody
to the second analyte. In one embodiment, the second analyte or fragment
thereof is a protein
or peptide. hi a further embodiment, the second analyte or fragment thereof
and a heavy
chain or light chain of the antibody reside on a single polypeptide chain. In
another
embodiment, the second analyte is carbonic anhydrase III and the first analyte
is myoglobin.
to In other embodiments, the second analyte is carbonic anhydrase III and the
first analyte is
fatty acid binding protein, myosin light chain or any other analyte released
from cardiac
tissue. The invention also embraces polynucleotides encoding a single-chain
polypeptide
comprising the immunoglobulin light or heavy chain and an analyte or fragment
thereof.
15 The invention is also directed to kits comprising some or all of the
various reagents
hereinbefore described in order to carry out any of the assays described and
variations
therefore embraced herein. Referring to the first analyte as analyte and the
second analyte as
marker, the following kits are embraced herein:
2o I~it I
1) a labeled binding partner to the analyte;
2) a conjugate between the marker and a second binding partner to the analyte;
and
3) an immobilized antibody to the maxker.
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Kit II
1) a labeled binding partner to the marker
2) a conjugate between the marker and a binding partner to the analyte; and
3) an immobilized second binding partner to the analyte.
s
Kit III
1) a labeled binding pautner to the analyte;
2) either
a) a second binding partner to the analyte conjugated to biotin, and
1o b) a conjugate between the marker and a biotin-binding reagent
or
c) a second binding partner to the analyte conjugated to a biotin-binding
reagent, and
d) a conjugate between the marker and biotin; and
is 3) an immobilized binding partner to the marker.
Kit IV
1) a labeled binding partner to the marker
2) either
2o a) a binding partner to the analyte conjugated to biotin and
b) a conjugate between the marker and a biotin-binding reagent
or
c) a binding partner to the analyte conjugated to a biotin-binding reagent,
and
2s d) a conjugate between the marker and biotin; and
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WO 02/16943 PCT/IBO1/01531
3) an immobilized second binding partner to the analyte.
In the foregoing kits, the binding partners are preferably antibodies or
binding portions
thereof, and both the binding partner to the analyte and the second binding
partner to the
analyte capable of simultaneously binding to the analyte. The conjugates
comprising the
marker may comprise an epitope of the marker. The immobilized binding partner
may be
provided in the form of a capture line on a test strip, or it may be in the
form of a microplate
well surface or plastic bead, by way of non-limiting examples. The kits may be
used in a
homogeneous format, wherein all reagents are added to the sample
simultaneously and no
i0 washing step is required for a readout, or the kits may be used in a mufti-
step procedure
where successive additions or steps are carried out, with the immobilized
reagent added last,
with an optional washing step. The teachings herein will allow a skilled
artisan to prepare
other variations in kit componentry and assay format which carry out the assay
of the
invention and variations fully embraced herein. Other reagents and
instructions may be
15 included with the foregoing reagents.
These and other aspects of the present invention will be better appreciated by
reference to the
following drawings and Detailed Description.
2o BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts schematically an assay of the invention, using a lateral flow
strip format, for
detecting cardiac-specific myoglobin by subtracting, from the detection of
myoglobin, any
carbonic anhydrase III (CAIII) present in the sample, which is co-released
with myoglobin
from non-cardiac sources. An. immobilized anti-myoglobin antibody is provided
at the
25 capture line, and two mobile conjugates, representing the analyte labeling
reagent, are
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CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
utilized: a detectable (gold-labeled) anti-CAIII antibody conjugate, and a
CAIII-anti-
myoglobin antibody conjugate.
Figure 2A depicts schematically another embodiment as in Figure 1, which
employs an
immobilized anti-myoglobin antibody at the capture line and, representing the
analyte
labeling reagent, three mobile conjugate reagents: a detectable (gold-labeled)
anti-CAIII
antibody conjugate, a CAIII-streptavidin conjugate, and a biotinylated anti-
myoglobin
antibody.
to Figure 2B depicts schematically another embodiment as in Figure 2A, but
using a
heterogeneous format.
Figure 3 depicts graphically the myoglobin, carbonic anhydrase III, and the
ratio
therebetween, in a series of myocardial infarction patients.
Figure 4 depicts graphically the myoglobin, carbonic anhydrase III, and the
ratio
therebetween, in a series of patients with muscle disease.
Figure 5 shows a depiction of an assay of the invention, with levels of the
components used
2o in modeling the signal needed to achieve the desired readout.
Figure 6 shows a theoretical analysis of the signal needed for various
combinations of levels
of myoglobin and carbonic anhydrase.
23
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WO 02/16943 PCT/IBO1/01531
Figure 7 shows the signal generated from a mathematical model of the present
assay over a
dose range of myoglobin with a myoglobin to carbonic anhydrase ratio of 2.9,
and a carbonic
anhydrase level of 2246 ng/ml.
Figure 8 shows the signal from a mathematical model of the present assay in
the presence of
a range of free carbonic anhydrase III, with a myoglobin:carbonic anhydrase
ratio of 2.9, and
6467 ng/ml of myoglobin.
Figure 9 shows the signal generated from a mathematical model of the present
assay from a
to ~ range of streptavidin-carbonic anhydrase III conjugate.
Figure 10 shows the signal generated from a mathematical model of the present
assay from a
range of biotinylated antibody levels.
15 Figure 11 shows the signal generated from a mathematical model of the
present assay from a
range of gold-conjugated antibody.
Figure 12 A-B depicts a single-chain polypeptide comprising carbonic anhydrase
III and
streptavidin (SEQ ID NO:1), joined by a two-amino-acid linker, which binds to
biotin and
2o also to an anti-carbonic anhydrase III antibody.
Figure 13 shows the electrophoretic purity of the streptavidin-carbonic
anhydrase III single-
chain polypeptide described in Figure 12.
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CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "preselected analyte" or "first analyte" refers to a
particular substance to be
measured in an assay of the invention and expressed as the difference between
or ratio over a
second analyte. In a particular embodiment, the preselected analyte may be a
protein that
may be released into a bodily fluid from a particular ("target") bodily source
under a certain
physiological or pathological condition desirous of being determined, and may
also be
released from another ("non-target") bodily source, the release therefrom
u~zrelated to the
physiological or pathological condition desirous of being determined, and thus
responsible
for obscuring the usefulness of the preselected analyte as a condition-
specific marker.
l0
"Non-target-bodily source" refers to the origin of a preselected analyte not
related to the
physiological of pathological condition being determined by the methods
herein.
"Non-target-bodily-source marker," also referred to simply as "marlcer" for
brevity, refers to
15 an analyte that is co-released with the preselected analyte into the bodily
fluid from sources)
other than the target source. It is also referred to as the "second analyte"
in the general
methods described herein. Thus, in the instance of the present method being
used
diagnostically for the human body, the level of the non-target-bodily-source
marker in the
bodily fluid is proportional to the level of preselected analyte not of the
origin desirous of
2o being assessed by the methods herein.
"Homogeneous" indicates that the assay, certain embodiments, is performed on
the sample in
a single step, as far as the user is concerned, without the need, as in
heterogeneous assays, for
adding reagents, washing or collecting intermediate samples, etc., and that a
positive readout,
CA 02420327 2003-02-24
WO 02/16943 . PCT/IBO1/01531
e, g., formation of color at the capture line, is indicative of a positive
result, i.e., that the
preselected analyte from the target source is present in the sample.
This application claims priority under 35 U.S.C. ~119(e) to Provisional
Applications serial
ntunbers 60/227,536, filed August 24, 2000, and 60/292,497, filed May 21,
2001, both of
which are incorporated by reference herein in their entireties.
The present assay is suitably applied using an immunoassay format which is
capable of
detecting the difference or ratio between levels of two or more analytes in a
biological sample
l0 and providing a single readout indicative of, for example, the level of one
analyte minus that
of the other. Depending on the relative affinities of the various reagents and
binding partners
used in the assay, the method can also readout the ratio among the analytes.
Such a readout is
useful when the level of a second analyte is needed to interpret the
diagnostic value of the
level of the first analyte. The first analyte must be present to obtain any
reading, but its level
15 is reduced by the level of the second analyte, and more particularly by
employing a
preselected analyte labeling reaction in which a binding partner for the
preselected analyte
and the second analyte compete for binding to the label.
This format may be used in several ways, one of which is to read out the ratio
of differences
2o in levels among two analytes, wherein the ratios are diagnostically useful,
such as but not
limited to total cholesterol and high-density lipoprotein (HDL) for assessing
risk of
atherosclerotic diseases. The format may also be used to identify the origin
or source of an
elevated level of a circulating analyte or marker, if more than one source is
possible, provided
release of the analyte from a second source is accompanied by release of
another marker
26
CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
specific to that second source. This may be performed qualitatively or
quantitatively by the
methods herein.
By way of example to place this invention in perspective, an example related
to the early and
accurate diagnosis of a heart attack is illustrated. Cardiac troponin I is a
heaxt-muscle-
specific marker which, if detected at elevated levels in circulation, is
absolutely diagnostic of
a heart attaclc. However, it is not present in detectable levels until about 6
hours following a
heart attaclc, and such a delay in accurate diagnosis after onset of chest
pain may delay the
administration of critical therapies, such as fibrinolytic therapy, to reduce
the morbidity and
to mortality of the disease. However, fibrinolytic therapy if administered to
an individual not
suffering from a heart attack may have serious complications (e.g.,
hemorrhage). Thus, early
and specific diagnosis is highly desirable.
In contrast to the specificity of the foregoing "late" marker troponin I,
following a heart
attack, the muscle damage marker myoglobin is detectable in circulation much
earlier than
troponin I, but myoglobin may also released from skeletal muscle following
skeletal muscle
injury. It thus is an ideal early heart attack maxker if its specific release
from heart (or lack of
specific release from skeletal muscle) can be assessed. However, elevated
myoglobin levels
could mean heart attack, skeletal muscle injury, or both. As noted above,
successful early
2o therapy (and lack of complications) is critically dependent on an accurate
diagnosis.
Simply measuring circulating myoglobin does not provide the needed diagnostic
accuracy,
since its source is not determinable. To determine whether an elevated level
of myoglobin is
of cardiac or non-cardiac origin, the single-readout assay of the present
invention takes
advantage of the simultaneous release from injured non-cardiac muscle of both
myoglobin
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CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
and caxbonic anhydrase III (CAIII). The latter is not released from heart
muscle as a
consequence of heart attack. Thus, separate assays for both myoglobin and
carbonic
anhydrase III could be used diagnostically to identify a heart attack, if the
health care
professional, when interpreting the results of these two assays, subtracts
from the myoglobin
value (representing this marlcer possibly derived from both cardiac and
skeletal sources) its
equivalent in carbonic anhydrase III level (taking into consideration the
relative ratio of
myoglobin to carbonic anhydrase III release from injured skeletal muscle).
This is a
complicated calculation. However, to simplify the calculation and
interpretation of these
results, an assay of the invention may quantitatively read out only the level
of myoglobin of
to cardiac origin, by essentially subtracting from the total myoglobin levels
the equivalent
amount of skeletal myoglobin present based on the amount of carbonic anhydrase
III in the
sample. Or in the instance of a qualitative assay, a positive signal may be
generated only if
the myoglobin level of cardiac origin is above a preestablished value
indicative of a heart
attack.
The methods of the invention are applicable to any combination of analytes,
preferably pairs
but it is not so limiting. Moreover, the relative amounts of the first and
second analyte and
the differentiating ability of the assay can be adjusted by selection of the
appropriate affiW ties
of the binding partners, preferably antibodies, of the various reagents of the
assay. Thus, if
2o the second analyte is released at very small amounts relative to the first
marker, by use of a
higher affinity antibody to the second analyte relative to the affinity of the
antibody to the
first analyte, in the various reagents of the invention, will have the effect
of giving more
weight to the level of the second analyte in reducing the readout of the
assay. These
variations can be readily determined to give a signal based on the relative
levels and cutoff
values of the analytes.
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WO 02/16943 PCT/IBO1/01531
A physiological or pathologic condition identifiable or diagnosable by
detecting the presence
in a bodily fluid of elevated levels of a marker or analyte that is specific
to the condition is
relatively easy to assess, as the mere presence of the analyte or increase
over normal levels is
indicative of the condition. Such analytes are usually unique to a particular
bodily source or
are only released from a single bodily source or site of the particular
condition. However,
many analytes often are not unique or specific to particular condition, and
the same analyte is
releasable from both the affected bodily source and possibly other sources,
confounding any
ascribing of the elevated levels to any one source. Thus, the present
invention offers a
l0 method for providing the necessary specificity to an assay for a particular
analyte by taking
advantage of the co-release from the non-target bodily source of another
analyte, herein
referred to as the non-target-bodily-source marker (or simply, "marker"),
which when present
in circulation indicates that the desired analyte is not wholly or in part
from the source of
interest. While a separate assay for the non-target-bodily-source marker could
be carried out
15 concurrently with the preselected analyte, with the diagnostician ignoring
or reducing the
presence or extent of the desired analyte based on the detected level of the
co-released
marker, such interpretation of multiple tests, particularly under emergency
conditions, is
tedious and subject to error. An assay has been devised herein which reads out
analyte level
only from the bodily source of interest, reducing or preventing a read-out if
the co-released
20 marker is present concurrently. As noted, this is achieved using an analyte
labeling reaction
that is inhibited by marker present in the sample. Thus, the assay behaves as
if the analyte is
indeed target-specific for the particular pathology, and eliminates any need
for the user to
correlate multiple positive and/or negative values with the outcome.
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WO 02/16943 PCT/IBO1/01531
In a further example of the methods herein, a method is provided for
determining with a~.l
assay the effective filtering capacity of a filter based on changes in pore
size. This is
applicable in vivo to measuring l~idney damage, as it is lcnown that adverse
changes in the
glomerulus, for example as a consequence of diabetic renal disease, lead to
lack of selective
filtering (and retention) of large molecules from the blood. With
deterioration, larger and
larger molecules fail to be retained by the lcidneys and appear in the urine.
Thus, in the
practice of the invention, a first analyte may be a small peptide molecule
known to pass
easily through the renal filtering system into the urine in the presence or
absence of kidney
disease. The second analyte may be a larger protein which in normally-
functioning kidneys
to is not excreted in the urine, but with increasing kidney damage, becomes
excreted to an ever-
increasing extent. In the absence of renal damage, the assay will detect the
level of the
peptide, indicating normal renal function. With increasing renal damage and
excretion of the
larger protein, the assay readout declines propoutional to the level of the
larger protein,
indicating a decline in renal function. Thus, a simple renal function test in
a test strip format
15 may be prepared following the teachings herein. In a specific embodiment,
the analyte pair
useful in the assessment of kidney damage is allcaline phosphatase, a normally-
secreted small
protein, and 5'-nucleotidase, a larger protein the appearance of which
indicates tissue
damage.
2o The following table describes some examples of the tests and reagents that
may be used to
carry out the assays of the invention. These are merely exemplary and non-
limiting as to the
choices of analytes and markers as well as the format in which the
differential assay of the
invention is performed.
CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
Test ImmobilizedLabeling Complex
Aualyte Marker reagent Capture Conjugate LabelisZg
Reage~
Myo CA3 MyoAbi (1) MyoAbZ:Bn SAV:CA3 CA3Ab* (2)
Myo CA3 MyoAbl MyoAb2:CA3 CA3Ab*
Myo CA3 CA3Ab1 - CA3:MyoAbl, MyoAb2*
or CA3:SAV
+
MyoAb 1:Bn
Myo FABP MyoAbl MyoAb2:Bn SAV:FABP FABPAb *(3)
~
Myo MLC MyoAbl MyoAb2:Bn SAV:MLC MLCAb* (4)
Alk-Phos5'-NTase Alk-PhosAblAlk-PhosAb2BnSAV:S'NTase 5'NTaseAb*
(5) (6)
LDL HDL LDLAbI LDLAb2:Bn SAV:HDL HDLAb * (7)
HDL LDL HDLAbI HDLAba:Bn SAV:LDL LDLAb* (8)
Key to abbreviations:
* = detectable label
Myo = myoglobin
Ab = antibody, with subscripts denoting antibodies that recog~uze different
epitopes of the
noted antigen to allow concomitant binding of two antibodies to that reagent
:Bn = conjugated biotin
SAV = streptavidin
CA3 = carbonic anhydrase III
to CA3: = CA3 conjugated to noted reagent
Key to footnotes:
(1) Numerous MyoAbs are available commercially, including for instance from
BioDesign
International of Saco, Maine, USA (2001 Catalog #s H86104M (IgGl), H86142M
(IgGl),
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WO 02/16943 PCT/IBO1/01531
H86423 (IgG2a), K3I013 (IgG2b); and from Spectral Diagnostics Inc. under
catalog
numbers MA-2010, MA-2040 & PM-1000.
(2) CA3Abs are available commercially, for instance from Spectral Diagnostics
under
catalog number MA-4010, and from BioDesign.
(3) FABP Abs are available for instance from BioDesign (cat #s H86101M (IgGl),
H86294M (IgG2b), and from Spectral Diagnostics under cat # MA-6010.
(4) MLC Abs can be obtained from Spectral Diagnostics under cat # 5010 or MLC1-
14.
(5) All-Phos Abs can be obtained from BioDesign under cat #s K45802M and
K45801M.
(6) 5'-NTase Abs can be raised against the enzyme which has the properties
reported by
1o Bachmann et al., Kidney International 1997 February; 51(2):479-82 and
references therein.
(7) HDL Abs can be obtained from numerous suppliers.
(8) LDL Abs can be obtained from numerous suppliers, including BioDesign (cat
#L62308G).
As mentioned above, these assays and connnercial sources of reagents for their
operation are
merely illustrative and non-limiting, and other assays and reagents may be
prepared in
accordance with the teachings herein.
To describe the general invention in another manner, let At represent the
first analyte in a
sample originating from the target source, and A" be the same analyte
originating from the
2o non-target source. Let B represent a second analyte which is released in
proportion to A"
only from the non-target source. The desirable measurement is At alone, yet
because they are
the same analyte, they cannot be distinguished as only total A can be
measured. However,
because the level of A" is proportional to the level of B in the sample, the
value of At can be
obtained using (Atplus A") minus B. The assay measures At plus A" but reduces
the value
with increasing levels of B in the sample.
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WO 02/16943 PCT/IBO1/01531
The method is applicable to any assay format, and is conveniently applied, in
a homogeneous
format, to a dry reagent test strip format. Such test strip formats generally
comprise a
membrane along which sample flows, piclcing up mobile reagents, possibly but
not
necessarily in succession, in a detection zone, and reading out test results
at a capture zone
where one or more mobile reagents and/or analyte are sequestered at a capture
line by an
antibody or other specific binding molecule for the analyte. Various bodily
fluids may be
used, such as but not limited to whole blood, plasma, serum, urine,
cerebrospinal fluid,
biopsy fluid, and tissue homogenates.
to
The relative ratio of release of both the analyte and the co-released maxker
from the non-
target source may be factored into the sensitivity and detectability of the
source-specific
analyte by adjusting the compositions of conjugates used in the assay.
Following from the
same example above, if the co-released marker is present at only a fraction of
the preselected
15 analyte released from the non-target bodily source, a decreased ratio of
carbonic anhydrase
III in the carbonic anhydrase III-containing conjugate of either assay format
will increase the
sensitivity of the assay to carbonic anhydrase III. The amount or ratios of
other components
may also be adjusted to adjust the sensitivity of the assay to provide either
the qualitative (yes
or no) or quantitative (value) readout. Such adjustments are fully embraced
herein.
The method of the invention may be carried out for any analyte for which a
marker is co-
released with the preselected analyte and is used to effectively subtract or
negatively
influence the level of preselected analyte of non-target bodily source origin
from the amalyte
from the target source, by the methods herein. Other uses include simply
expressing ratios or
differences in the levels of two analytes. While several analytes and markers
are known, the
33
CA 02420327 2003-02-24
WO 02/16943 PCT/IBO1/01531
methods herein offer the development of a large number of new and specific
diagnostic tests,
using either heterogeneous or homogeneous formats, that do not require
sophisticated
analysis or interaction by user or computer of the results. Methods for
simultaneously
measuring the level of numerous analytes in a blood sample, e.g., biological
"chips" with
sites for a plurality of binding sites for biomolecules, such devices
requiring computational
analysis of the results and integration of data to provide the results. More
simply, individual
assays for the preselected analyte and the non-target-bodily-source marker may
be performed,
the results require mental (or mechanical) integration of the results to
arrive at the answer.
For example, test strip assay for myoglobin and carbonic anhydrase III may be
performed. If
l0 myoglobin is elevated, and carbonic anhydrase III is elevated, the origin
of circulating
myoglobin is likely of non-cardiac oxygen. If carbonic anhydrase III is
absent, the
myoglobin is likely of cardiac origin. However, such tests require additional
reagents,
additional samples, assays under similar conditions, and above all, an
interpretation of the
results of multiple tests to arrive at a conclusion. In contrast, the instant
methods are
15 homogeneous, single-test assays that directly read out the desired results.
Thus, the methods
herein embrace a homogeneous source-specific-assay which detects any analyte.
In
alternative embodiments, heterogeneous assays are provided which serve the
same purposes.
The examples described in detail herein are merely exemplary.
2o While a preferred format for the methods herein are membrane-based test
strips which may
be used, for example, a whole blood obtained by finger puncture in an
emergency room or
other point-of care site, the methods are adaptable to any manual, semi-
automated or
automated measurement method including but not limited to a single-use test
strip read by
eye or reflectometer, an automated assay analyzer capable of processing
numerous samples, a
25 multiple-analyte test strip, etc. Microtiter plate-based or microbead-based
assays are also
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adaptable for the assay herein, where the immobilized reagent is bound to a
microtiter plate
well or to a plastic bead. Membrane-based test strips which utilize whole
blood are known in
the art.
Sources of sample for the assay herein is preferably a biological sample such
as whole blood,
plasma, serum, urine, cerebrospinal fluid, biopsy specimens, etc., but is not
limited thereto
and may extend to other sources in which the level of a particular analyte and
analyte source
of interest may me marked by the same analyte from another source, and the
level of analyte
from the other source may be gauged by another co-released or coordinately
present marker
to in the sample. Such uses may include food processing and analysis,
wastewater,
environmental analysis, etc., where the origin of a particular analyte is
determinable. By way
of illustration only, a test to determine the level, if any, in a reservoir of
a particular
enteropathogenic coliform bacterial contaminant originating from runoff from a
dairy farm
may be determined even though the same coliform may originate from the
occasional squirrel
15 excrement falling in the reservoir. A test as described herein which
detects the coliform but
"subtracts" therefrom a squirrel intestine-specific bacterium yields the level
of the cattle-
derived contaminant.
Thus, an assay format is preferred, in which binding partners such as
antibodies can be
2o obtained or prepared for the analytes. As noted, biotin-avidin, biotin-
streptavidin or other
biotin-binding-reagent reactions can be used to enhance or modulate the test.
However, any
such assay can be devised using other binding partners to the analyte and
marker, including
but not limited to extracellular or intracellular receptor proteins which
recognize the analytes,
binding fragments thereof, hybridization probes for nucleic acids, lectins for
carbohydrates,
25 etc. The particular selection of binding partners is not limiting, provided
that the binding
CA 02420327 2003-02-24
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partners permit the test to operate as described herein. As noted above, the
preselected
analyte, when present, is detectable by binding by two binding partners, one
immobilized on
the test strip (or whatever format the assay is provided) and another part of
a conjugate. This
is taken into consideration in the selection of the reagents for the assay.
The dry test strip may be set up in any format in which contact of the sample
with the
reagents is permitted and the formation and mobility of the immunocomplexes
and other
complexes forming therein are permitted to flow a~ld contact an immobilized
reagent at the
capture line. Various format are available to achieve this purpose, which may
be selected by
l0 the skilled artisan.
The label portion of the mobile, labeled antibody to the non-target-bodily-
source marker may
be a visible label, such as gold or latex, an ultraviolet absorptive marker,
fluorescent marker,
radionuclide or radioisotope-containing marker, an enzymatic marker, or any
other detectable
15 label. A visibly detectable marker or one that can be easily read in a
reflectometer is
preferred, for use by eye, reading or confirmation with a reflectometer. Other
labels may be
applicable to other semi-automated or automated instrumentation.
The conjugates of the invention may be prepared by conventional methods, such
as by
20 activation of an active moiety, use of homobifunctional or
heterobifunctional cross-linking
reagents, carbodiimides, and others known in the art. Preparation of, for
example, a gold-
labeled antibody, a conjugate between an antibody and an analyte (not an
immunocomplex
but a covalent attachment which allows each member to independently exhibit
its binding
properties), biotinylation of an antibody, conjugation of streptavidin with a
protein,
25 immobilization of antibodies on membrane surfaces, etc., are all methods
known to one of
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skill in the art. The preparation of the reagents of the methods herein extend
to, for example,
recombinant expression of a single-chain polypeptide comprising streptavidin
or the biotin
binding site of streptavidin or another biotin-binding molecule and at least
the epitope of the
non-target-bodily-source marker recognized by the antibody to the latter.
Thus, a
polynucleotide which comprises the coding sequence for each portion,
optionally linlced by a
segment encoding a polypeptide spacer or linker portion, is expressed such
that both
members on the single-chain polypeptide retain their desired activities in the
assay. The
invention extends to such single-chain polypeptide reagents useful for the
assays described
herein, as well as polynucleotides encoding the single-chain polypeptides, as
well as
' expression vectors, microorganisms containing such vectors and related means
for expressing
such polynucleotides to provide the desired single-chain polypeptides. In
another example,
recombinant preparation of immunoglobulin molecules in which the polypeptide
marker is
present in a single-chain polypeptide with either the heavy chain or light
chain of the
immunoglobulin are embraced herein, such that the assembled immunoglobulin
molecule (or
fragment) comprises the antigen binding sites) as well as epitopes of the
marker necessary
for the operation of the hereindescribed assay. Polynucleotides expressing
such polypeptides,
microorganisms and eukaryotic cells expressing and producing such products
including
antibodies wluch comprise a single-chain component with the marker, and other
means for
preparing such reagents are embraced herein. In a preferred embodiment, the
marker
polypeptide is fused at the C-terminus of the heavy chain of the particular
immunoglobulin
class and subclass desired. Moreover, other binding parhlers for the
preselected analyte other
thm an antibody, such as a receptor, may be provided in a conjugate with the
marlcer in a
single-chain or comprising a single-chain polypeptide with the desired
activity of both
components. Such methods to facilitate the preparation or parameters of the
reagents herein,
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as well as considerations such as ratios between the components, binding
affinities, color
density, etc., are embraced herein.
One example of such a single-chain polypeptide is described as SEQ ID NO:l, an
expression
product of a polynucleotide (SEQ ID N0:2) comprising streptavidin and carbonic
anhydrase
III. The present invention is also directed to this and related compositions.
It also extends to
polynucleotides encoding the single-chain polypeptides of the invention, such
as all of those
which encode SEQ ID N0:1. The polynucleotide sequences embraced herein include
degenerate variants which encode the same polypeptide sequences, by virtue of
the
to degeneracy of the genetic code, as well as variants in the polynucleotide
sequences which
result in altered amino acid sequences but are not of consequence in the
properties of the
expressed hybrid polypeptide.
As noted in the Summary above, the assays of the invention may be provided in
two
15 orientations: in one, the labeled reagent is a binding partner to the
analyte and the
immobilized reagent is a binding partner to the marker; in an alternate
embodiment, the
labeled reagent is a binding partner to the marker and the immobilized reagent
is a binding
partner to the analyte. In both assays, a conjugate between the marker and a
binding partner
to the analyte is used; in the alternate format of either assay with slightly
different reagents,
2o in place of the conjugate between the marker and a binding partner to the
analyte, the reagent
pairs used may be either 1) a binding partner to the analyte conjugated to
biotin, and a
conjugate of the marlcer and a biotin-binding reagent such as streptavidin, or
2) a binding
partner to the analyte conjugated to a biotin-binding molecule such as
streptavidin, and a
conjugate of the marlcer and biotin. Both orientations and reagent selections
are fully
25 embraced herein and may be selected depending on the desired sensitivity of
the assay, the
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characteristics of the reagents, the relative ratios of analyte to marker
encountered, and other
parameters.
The invention is also directed to kits comprising some or all of the various
reagents
hereinbefore described in order to carry out any of the assays described and
variations thereof
embraced herein. A lcit may have at least one reagent for carrying out an
assay of the
invention, such as a kit comprising a conjugate between a biotin-binding
reagent and a
marker, such as a single-chain polypeptide comprising streptavidin and a
polypeptide marker.
Preferably, the kit comprises all of the reagents needed to carry out any one
of the
to aforementioned assays, whether it be homogeneous, heterogeneous, comprise a
single
conjugate of the marker conjugated to an antibody to the analyte, or comprise
two reagents
which serve this function (such as a biotinylated antibody to the analyte plus
a streptavidin-
marker conjugate, or a biotinylated marker plus a streptavidin conjugated to
an antibody to
the analyte conjugate), or whether the assay employs an immobilized antibody
to the analyte
15 and a labeled antibody to the marker, or an immobilized antibody to the
marker and a labeled
antibody to the analyte. Referring to the first analyte as analyte and the
second analyte as
marker, and a second binding partner as a binding partner which recognizes a
different
epitope tha~.i the first binding partner mentioned, the following kits are non-
limiting examples
of those embraced herein:
a
Kit I
1) a labeled binding partner to the analyte;
2,) a conjugate between the marker and a second binding partner to the
analyte;
and
3) an irrunobilized antibody to the marker.
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Kit II
1) a labeled binding partner to the marker;
2) a conjugate between the marker and a binding partner to the analyte; and
3) an immobilized second binding partner to the analyte.
Kit III
1) a labeled binding partner to the analyte;
2) a second binding partner to the analyte conjugated to biotin;
1o 3) a conjugate between the marker and a biotin-binding reagent; and
4) an immobilized binding partner to the marker.
Kit IV
1) a labeled binding partner to the analyte;
2) a second binding partner to the analyte conjugated
to a biotin-binding reagent;
3) a conjugate between the marker and biotin; and
4) an immobilized binding partner to the marker.
I~it V
2o 1) a labeled binding partner to the marker;
2) a binding partner to the analyte conjugated
to biotin;
3) a conjugate between the marker and a biotin-binding
reagent; and
4) an immobilized second binding partner to the
analyte.
CA 02420327 2003-02-24
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Kit VI
1) a labeled binding partner to the marker;
2) a binding partner to the analyte conjugated to a biotin-binding reagent;
3) a conjugate between the marlcer and biotin; and
4) an immobilized second binding partner to the analyte.
Kit VII
1) a conjugate between the marker and a binding partner to the analyte.
1 o Kit VIII
1) a second binding partner to the analyte conjugated to biotin, and
2) a conjugate between the marker and a biotin-binding reagent.
Kit I~
1) a binding partner to the analyte conjugated to a biotin-binding reagent,
and
2) a conjugate between the marker and biotin.
In any of the foregoing kits, the binding partners are preferably antibodies
or binding portions
thereof, and both the binding partner to the analyte and the second binding
partner to the
2o analyte capable of simultaneously binding to the analyte. The conjugates
comprising the
marker may comprise an epitope of the marker, such that it is recognized by a
binding partner
to the marker. The conjugates of the kits, if both members are independently
peptides or
polypeptides, may be in the form of a single-chain polypeptide comprising both
members,
each exhibiting its activity independently in the single-chain polypeptide.
The immobilized
binding partner may be provided in the form of a capture line on a test strip,
or it may be in
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CA 02420327 2003-02-24
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the form of a microplate well surface or plastic bead, by way of non-limiting
examples of
immobilized carriers for binding partners. As mentioned above, the kits with
or without
immobilized reagent may be used in a homogeneous format, wherein all reagents
are added
to the sample simultaneously and no washing step is required for a readout, or
the kits may be
used in a multi-step procedure where successive additions or steps are carried
out, with an
optional washing step or transfer of components from one container to another.
The
teachings herein will allow a skilled artisan to prepare other variations in
kit componentry
and assay format which carry out the assay of the invention and its variations
fully embraced
herein. Other reagents, containers, and instructions may be included with any
of the
to foregoing bits.
By way of non-limiting example, and following the above example of an assay
for detecting
myoglobin released from cardiac tissue and reducing it by the level of CAIII
that would be
co-released with myoglobin from skeletal muscle, the foregoing examples of
kits comprise
15 the following reagents in a preferred embodiment:
Kit I
1) a labeled antibody to myoglobin;
2) a conjugate between CAIII and a second antibody to myoglobin; and
2o 3) an immobilized antibody to CAIII.
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I~it II
1) a labeled antibody to CAIII;
2) a conjugate between CAIII and an antibody to myoglobin; and
3) an immobilized second antibody to myoglobin.
I~it III
1) a labeled antibody to myoglobin;
2) a second antibody to myoglobin conjugated to biotin;
3) a conjugate between CAIII and streptavidin; and
1 o 4) an immobilized antibody to CAIII.
I~it IV
1) a labeled antibody to myoglobin;
2) a second antibody to myoglobin conjugated to streptavidin;
3) a conjugate between the CAIII and biotin; and
4) an immobilized antibody to CAIII.
I~it V
1) a labeled antibody to CAIII;
2o 2) an antibody to myoglobin conjugated
to biotin;
3) a conjugate between CAIII and streptavidin;
and
4) an immobilized second antibody to
myoglobin.
Kit VI
1) a labeled antibody to CAIII;
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2) an antibody to myoglobin conjugated to streptavidin;
3) a conjugate between CAIII and biotin; and
4) an immobilized second antibody to myoglobin.
Kit VII
1) a conjugate between CAIII and an antibody to myoglobin.
Kit VIII
1) a second antibody to myoglobin conjugated to biotin, and
2) a conjugate between CAIII and streptavidin.
Kit IX
1) an antibody to myoglobin conjugated to streptavidin, and
2) a conjugate between CAIII and biotin.
Of course, the conjugate between CAIII and streptavidin may be a single-chain
polynucleotide comprising CAIII or an anti-CAIII-antibody-binding fragment
thereof, and
streptavidin or a biotin-binding fragment thereof. The conjugate between CAIII
and an
antibody to myoglobin may comprise a single-chain polypeptide of CAIII or an
epitope
2o thereof and a light or heavy immunoglobulin chain, which is than assembled
to provide a
hybrid molecule of an anti-myoglobin antibody and CAIII.
Moreover, a lit of the invention may comprise a polynucleotide encoding a
single-chain
polypeptide comprising CAIII and streptavidin.
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The foregoing exalnples of lcits for detecting cardiac myoglobin in a bodily
fluid are merely
exemplary of tlus embodiment, and as mentioned above, the reagents may be
tailored to
measure a large number of other analytes and markers for various purposes.
The present invention may be better understood by reference to the following
non-limiting
Examples, which are provided as exemplary of the invention. The following
examples are
presented in order to more fully illustrate the preferred embodiments of the
invention. They
should in no way be construed, however, as limiting the broad scope of the
invention.
to Example 1
Two-conjugate, qualitative homogeneous assay for heart attack
Figure 1 illustrates the design and operation of a membrane strip-format assay
which is
positive for myoglobin only if carbonic anhydrase III is absent. Present in
the detection zone
of the test strip is analyte labeling reagent in the form of two mobile
reagents: 1) a gold-
15 labeled, affinity-purified polyclonal IgG antibody to carbonic anhydrase
III, and 2) a
conjugate between CAIII and an anti-myoglobin monoclonal IgG antibody,
prepared using a
heterobifunctional cross-linking reagent. hnmobilized at the capture line is
another anti-
myoglobin monoclonal IgG antibody, recognizing a different epitope on
myoglobin than that
of the aforementioned conjugate. The assay format may be as described in co-
pending
2o application serial number 09/130,164, filed August 6, 1998, now U.S. Patent
6,171,870, in
which a whole blood sample is applied to the device and red blood cells in the
whole blood
sample are detained in migration providing a red-cell-free plasma front at the
capture line for
visualization. A 25 microliter sample of whole blood from a patient in the
emergency room
presenting with chest pain of a few hours' onset, and having a history of poor
dietary habits
25 and sedentary life style, is applied to the device, and the results are
read after the test
CA 02420327 2003-02-24
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complete window indicates the test is complete. During the flow of sample, any
myoglobin
in the sample forms an immunocomplex with the mobile conjugate comprising the
anti-
myoglobin antibody and carbonic anllydrase III, and the myoglobin
immunocomplexed
therewith binds to the immobilized anti-myoglobin antibody. The gold-labeled
anti-carbonic
anhydrase III reagent binds to the carbonic anhydrase III in the first
immunocomplex,
forming a colored band at the capture line and indicating the presence of
myoglobin in the
absence of carbonic anhydrase III. This result is diagnostic of a heart
attack. Treatment with
fibrinolytic therapy is indicated.
to In another example, a sample is obtained by emergency medical technicians
at the site of a
traffic accident in which an individual with a similar life style as above but
with chest pain of
onset immediately following the chest striking the automobile steering wheel.
The test as
above is used. In this case, no colored band forms at the capture line,
indicating that if
myoglobin was released as a consequence of skeletal trauma in the accident,
its level is
15 compensated (i.e., reduced) by the simultaneous release of carbonic
anhydrase III. The
reaction chain comprising the carbonic anhydrase III-anti-myoglobin antibody,
myoglobin,
and the immobilized anti-myoglobin antibody forms are a consequence of
slceletal muscle
myoglobin present in the sample, but the coincident presence of carbonic
anhydrase III binds
to the labeled anti-carbonic anhydrase III antibody and thus this labeled
reagent is not
Zo available to bind to the carbonic anhydrase III-anti-myoglobin antibody
conjugate at the
capture line. A heart attack is ruled out. The patient is treated for chest
trauma.
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Example 2
Three-conjugate, qualitative and quantitative assays for heart attack
Figure 2A illustrates the design and operation of a membrane strip-format
assay which is
positive for myoglobin only if carbonic anhydrase III is absent. Present in
the detection zone
of the test strip is an analyte labeling reagent in the form of three mobile
reagents: 1) a gold-
labeled, affinity-purified polyclonal IgG antibody to carbonic anhydrase III,
prepared as
described in Example 1 above, 2) a conjugate between carbonic anhydrase III
and
streptavidin, prepared either by engineering a single-chain polypeptide
comprising carbonic
anhydrase III and streptavidin, or using a heterobifunctional cross-linking
agent to cross-link
l0 the members, and 3) a biotinylated anti-myoglobin monoclonal IgG antibody.
Immobilized
at the capture line is another anti-myoglobin monoclonal IgG antibody,
recognizing a
different epitope on myoglobin than that in the biotinylated reagent. The
assay format may
be as described in US patent 6,171,870, incorporated herein by reference in
its entirety, in
which a whole blood sample is applied to the device and red blood cells in the
whole blood
15 sample are detained in migration providing a red-cell-free plasma front at
the capture line for
visualization. A 25 microliter sample of whole blood from a patient in the
emergency room
presenting with chest pain of a few hours onset, and a history of poor dietary
habits and
sedentary life style, is applied to the device, and the results are read after
the test complete
window indicates the test is completed. During the flow of sample, any
myoglobin in the
2o sample forms an immunocomplex with the mobile, biotinylated anti-myoglobin
antibody and
the immunocomplex binds to the immobilized anti-myoglobin antibody at the
capture line.
The gold-labeled anti-carbonic anhydrase III reagent binds to the streptavidin-
carbonic
anhydrase III conjugate, and the streptavidin binds to the now-immobilized
biotinylated anti-
myoglobin antibody, producing a colored band at the capture line. This result
is diagnostic of
25 a heart attack. The patient is treated immediately with fibrinolytic
therapy.
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Similarly, and as shown in Figure 2B, the same assay depicted in Figure 2A and
described
above can be performed using a heterogeneous format, requiring a wash step
between
incubation of sample with reagents, and detection of label, in order to remove
unbound label
from the reaction mix. This quantitative assay is carried out according to the
following
procedure.
1. Coating: Coat wells with 100 microliters of rabbit anti-myoglobin (Spectral
Diagnostics
Inc.) at a concentration of 5 micrograms per milliliter in 50 mM
carbonate/bicarbonate
to Coating Buffer, pH 9.6 (always prepare in glass). Seal the plate with plate
sealer and
store at 4 C ovenught.
2. Washing: Wash plates 3 times with PBS-0.05% Tween 20 and 1 time with
ultrafiltered
(UF) water.
3. Blocking: Block the wells with 200 microliters of 1 % digested casein at
room
temperature for 1 hour while shaking at 400 rpm.
4. Repeat washing step 2.
5. Add 50 microliters of sample per well.
6. Add 25 microliters/well of diluent buffer (PBS containing 0.005% Tween 20
and 0.25%
BSA) containing 4 micrograms per milliliter of anti-CAIII mAb 2CA-4 (Spectral
2o Diagnostics Inc.) conjugated with HRP.
7: Incubate for 10 min at room temperature with shaking.
8. Add 25 microliters/well of diluent buffer containing 8 micrograms per
milliliter of
biotinylated anti-myoglobin mAb 2mb-295 (Spectral Diagnostics Inc.) and 2
micrograms
per milliliter of streptavidin-CAIII recombinant protein (Spectral Diagnostics
Inc.).
9. Incubate for 30 min at room temperature with shaking.
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CA 02420327 2003-02-24
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r
10. Repeat washing step 2.
11. Add 100 microliters/well of substrate solution (1 OPD tablet, 12.5 ml of
phosphate/citrate
buffer, pH 5.0, 125 microliters of 3% H20Z).
12. Incubate for 30 min at room temperature in the dark.
13. Stop reaction with 50 microliters/well of 4 N H2S04 and read at 490 nm in
a plate reader.
The results fiom the plate reader are then compared with a standard curve to
calculate cardiac
myoglobin concentration. A standard curve is generated with the same method
using a set of
calibrators.
The foregoing details of the procedure are merely exemplary and many
variations in its
details, such as the conditions for coating, reaction, various concentrations
of reagents,
buffers, etc., may be varied and remain with the scope of the present
invention.
In another example, a sample is obtained by emergency medical technicians at
the site of a
traffic accident in Which an individual with a similar life style as above but
with chest pain of
onset immediately following the chest striking the automobile steering wheel.
The test as
above is used. In this case, no colored band forms at the capture line,
indicating that if
myoglobin was released as a consequence of skeletal trauma in the accident,
its level is
2o compensated (i.e., reduced) by the simultaneous release of carbonic
anhydrase III. In this
case, in the presence of both myoglobin and carbonic anhydrase III, the
myoglobin in the
sample permits the binding of the carbonic anhydrase III-streptavidin
conjugate to the
biotinylated anti-myoglobin antibody and the latter to myoglobin on the
immobilized ~anti-
myoglobin antibody, but the carbonic anhydrase III present in the sample binds
to the gold-
labeled anti-carbonic anhydrase III antibody, making it no longer available to
bind to the
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1 carbonic anhydrase III-streptavidin conjugate, and no color is deposited at
the capture line. A
heart attack is ruled out. The patient is treated for trauma to the chest, and
not with
potentially dangerous fibrinolytic therapy.
Example 3
Measurement of myoglobin of cardiac origin
Before preparing the reagents and assay format for a homogeneous test to
measure the level
of myoglobin of cardiac origin, the parameters under which the assay should
operate were
developed using mathematical models. In this assay, the level of total
myoglobin in a sample
to of blood is subtracted by the level of myoglobin of skeletal origin, the
latter determined based
on the detection of carbonic anhydrase III, which is co-released with
myoglobin from skeletal
muscle tissue, but is not released from cardiac tissue. The test parameters
are established
such that a positive test indicates a sufficiently high amount of myoglobin of
cardiac origin is
present to diagnose a heart attaclc. The assay system employed is that
described in Example
15 2, above.
To establish the cut-off value between a myoglobin-carbonic anhydrase III
differential
diagnostic of a heart attack versus that indicative of skeletal muscle damage,
actual patient
data from a series of myocardial infarct patients and a series of skeletal
muscle damage
2o patients were plotted (from highest to lowest myoglobin level) along with
the ratio between
the markers (Figures 3 and 4, respectively). The myocardial infarct patient
mean ratio was
40.6, with a range of 4.6 to 170; that of the skeletal muscle patients was
2.3, with a range of
0.56 to 5.6. A ratio of 2.9 was selected as the cutoff for diagnosis of a
suspected heart attack.
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A model system with the necessary reagents was set up, according to Figure 5.
The
detectable signal achieved by the assay at various levels of carbonic
anhydrase III and
myoglobin are shown in Figure 6, with a horizontal line drawn at a signal
level of 4.6 for
heart attaclc patients (and 5.06 for non-heart attack patients), the
detectable cut-off level.
Using a myoglobin:carboiuc anhydrase ratio of 2.9 and a free carbonic
anhydrase level of
2246 nglml (8.02 x 10-8 M), the signal generated over a range of myoglobin
levels is shown
in Figure 7. Likewise, in Figure 8, the signal generated over a range of
carbonic anhydrase
values for a ratio of 2.9 and a free myoglobin level of 6467 nghnl (3.59 x 10-
7 M) is shown.
to Figure 9 depicts the effect of different amount of the streptavidin-
carbonic anhydrase III
(SAV-CAIII) conjugate, and Figure 10 the amount of biotinylated mobile
antibody. Figure
11 shows the signal for a range of gold-labeled antibody.
Example 4
15 Preparation of a single-chain streptavidin-carbonic anhydrase III
polypeptide
(SAV-CAIII)
In order to establish a model system, a single-chain polypeptide comprising
streptavidin and
carbonic anhydrase III ("SAV-CAIII") was prepared and expressed. SAV and CAIII
were
linked by insertion of a restriction site at the C-terminus of SAV and N-
terminus of CAIII.
2o This site resulted in an addition of two extra amino acid residues, Thr and
Arg, between SAV
and CAIII as indicated in SEQ ID NO:1 and Figure 12A-B. This polynucleotide
(SEQ ID
NO:2) was inserted into pet expression vector and expressed in E. coli.
The polypeptide retained biotin-binding affinity as well as recognition by
anti-carbonic
25 anhydrase III antibodies. It was electrophoretically pure (Figure 13). A
test utilizing a gold-
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labeled anti-carbonic anhydrase polyclonal antibody (2CA-4) at 60
micrograms/ml, the
foregoing SAV-CAIII conjugate at 27.6 micrograms/ml, and a biotinylated
monoclonal anti-
troponin I antibody were combined. Using a BIAcore device to measure extent of
complex
formation, the foregoing combination generated 211 relative units. However,
when wild-type
streptavidin was used in place of the single-chain polypeptide, the value was
10 relative units.
And in the absence of the single-chain polypeptide, the value was 11 relative
units. This
preliminary study establishes the capability of the biotinylated antibody to
bind the
streptavidin-carbonic anhydrase III single-chain polypeptide, which in turn is
capable of
binding the gold-labeled anti-carbonic anhydrase III antibody.
to
Example 5
Assay of the invention
In a demonstration of the operation of the assay of the invention, the
following reagents were
used. A rabbit anti-myoglobin capture antibody was irmnobilized to a surface.
The mobile
15 reagents included 1) a biotinylated anti-myoglobin antibody (2MB-295); 2)
the above-
mentioned SAV-CAIII single-chain polypeptide; and 3) gold-labeled anti-
myoglobin
antibody (2CA-4). A strip-type test was performed with 500 ng/ml myoglobin and
another
with 500 ng/ml myoglobin and 500 ng/ml carbonic anhydrase III. The former gave
a value of
0.155, and the latter 0.086. This illustrates the achievement of an object of
the invention.
The present invention is not to be limited in scope by the specific
embodiments describe
herein. Indeed, various modifications of the invention in addition to those
described herein
will become apparent to those spilled in the art from the foregoing
description and the
accompanying figures. Such modifications are intended to fall within the scope
of the
appended claims.
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Various publications are cited herein, the disclosures of which are
incorporated herein by
reference in their entireties.
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