U.S. patent application number 11/751537 was filed with the patent office on 2008-12-18 for differential immunoassay.
This patent application is currently assigned to Nanogen, Inc.. Invention is credited to Qinwei Shi.
Application Number | 20080311591 11/751537 |
Document ID | / |
Family ID | 27397733 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080311591 |
Kind Code |
A1 |
Shi; Qinwei |
December 18, 2008 |
Differential Immunoassay
Abstract
The invention provides assay methods and kits that in general
measure the level of a first analyte in a sample reduced by the
level of a second analyte present in the same sample. In one
embodiment, where levels of a first analyte from a first source is
desirably determined and first analyte in the sample released from
a second source is accompanied by proportional co-release of a
second analyte, the assay identifies the level of first analyte
released only from the first source. For analytes within bodily
fluids, the assay can differentiate between elevated levels of
analyte specific to the particular physiological or pathological
state and elevated levels not specific to the particular state,
providing single tests with diagnostic utility.
Inventors: |
Shi; Qinwei; (Etobicoke,
CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
One Market, Spear Street Tower, Suite 2800
San Francisco
CA
94105
US
|
Assignee: |
Nanogen, Inc.
San Diego
CA
|
Family ID: |
27397733 |
Appl. No.: |
11/751537 |
Filed: |
May 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10673781 |
Sep 29, 2003 |
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11751537 |
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09938270 |
Aug 23, 2001 |
6673562 |
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10673781 |
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60292497 |
May 21, 2001 |
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60227536 |
Aug 24, 2000 |
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Current U.S.
Class: |
435/7.4 ;
436/518 |
Current CPC
Class: |
G01N 33/532 20130101;
Y10S 436/819 20130101; G01N 33/6887 20130101; Y10S 435/972
20130101; Y10S 435/81 20130101; G01N 33/542 20130101; G01N 33/581
20130101; G01N 33/54306 20130101; G01N 33/573 20130101 |
Class at
Publication: |
435/7.4 ;
436/518 |
International
Class: |
G01N 33/573 20060101
G01N033/573; G01N 33/543 20060101 G01N033/543 |
Claims
1-48. (canceled)
49. A method of detecting the presence of a first analyte in a
sample, said method comprising: contacting the sample with a
conjugate comprising an epitope of the second analyte and a binding
portion that recognizes a first epitope of the first analyte to
form a labeled complex comprising the first analyte and the
conjugate; and detecting the presence of the labeled complex,
wherein when a second analyte is present in the sample, the
formation of the labeled complex is inhibited and wherein the
presence of the labeled complex indicates the presence of the first
analyte in the sample.
50. The method of claim 49, wherein said contacting comprises:
contacting the sample with a) the conjugate, and b) a mobile,
labeled first binding partner that recognizes an epitope of the
second analyte to form a reaction mixture; and contacting the
reaction mixture with an immobilized second binding partner that
recognizes a second epitope of the first analyte to form a labeled
complex, wherein said labeled complex comprises the immobilized
third binding partner, the first analyte, the conjugate, and the
mobile, labeled second binding partner, and wherein said first
analyte and second analyte are distinct from one another, said
binding portion does not recognize the second analyte, and said
first binding partner does not recognize the first analyte.
51. The method of claim 50, wherein the conjugate comprises a
full-length second analyte.
52. The method of claim 50, wherein the binding portion and the
first and second binding partners are antibodies.
53. The method of claim 50, wherein the first epitope and second
epitope are distinct binding sites on the first analyte.
54. The method of claim 50, wherein the epitope of the second
analyte and the binding portion are covalently linked.
55. The method of claim 50, wherein the conjugate is a single-chain
polypeptide that comprises the epitope of the second analyte and
the binding portion in operable form.
56. The method of claim 55, wherein the conjugate comprises the
amino acid sequence of SEQ ID NO: 1.
57. The method of claim 50, wherein the conjugate further comprises
a binding interface between the epitope of the second analyte and
the binding portion, said binding interface comprising biotin and
streptavidin respectively linked to: the epitope of the second
analyte and the binding portion, or the binding portion and the
epitope of the second analyte.
58. The method of claim 50, wherein the first analyte is a cardiac
analyte.
59. The method of claim 58, wherein the cardiac analyte is
myoglobin and the second analyte is released from a non-cardiac
source.
60. The method of claim 59, wherein the second analyte is carbonic
anhydrase III
61. The method of claim 50, comprising contacting the sample first
with the mobile, labeled first binding partner then the
conjugate.
62. A method of diagnosing a condition in a subject, said method
comprising: contacting a sample from the subject with a conjugate
comprising an epitope of a second analyte and a binding portion
that recognizes a first epitope of a first analyte to form a
labeled complex comprising the first analyte and the conjugate; and
detecting the presence of the labeled complex, wherein when the
second analyte is present in the sample, the formation of the
labeled complex is inhibited and wherein the presence of the
labeled complex is indicative of the condition in the subject.
63. The method of claim 62, wherein the condition being diagnosed
is a heart attack, the first analyte is myoglobin, and the second
analyte is carbonic anhydrase III.
64. The method of claim 62, wherein the condition being diagnosed
is a heart attack, the first analyte is fatty acid binding protein,
and the second analyte is carbonic anhydrase III.
65. The method of claim 62, wherein the condition being diagnosed
is risk for atherosclerotic disease, and the first analyte is total
cholesterol and the second analyte is high-density lipoprotein.
66. A method of detecting analyte from a first source, said method
comprising: contacting a sample with a conjugate comprising an
epitope of a marker from a second source and a binding portion that
recognizes a first epitope of the analyte to form a labeled complex
comprising the analyte and the conjugate; and detecting the
presence of the labeled complex, wherein the first source and
second source are distinct from one another, and when the marker is
present in the sample, the formation of the labeled complex is
inhibited, and wherein the presence of the labeled complex is
indicative of the presence of analyte from the first source.
67. The method of claim 66, wherein the marker is selected to be a
biomolecule that is released from the second source in proportion
to the level of analyte released from the second source.
68. The method of claim 66, wherein the analyte is selected to be a
biomolecule that can originate from the first source and the second
source.
69. The method of claim 66, wherein the first source is cardiac
tissue and the second source is muscle tissue.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/673,781, filed Sep. 29, 2003; which is a
divisional of U.S. patent application Ser. No. 09/938,270, filed
Aug. 23, 2001, now issued as U.S. Pat. No. 6,673,562; which in turn
claims the benefit of U.S. provisional application Ser. No.
60/292,497, filed May 21, 2001, and U.S. provisional application
Ser. No. 60/227,536, filed Aug. 24, 2000 under 35 U.S.C.
.sctn.119(e), the contents of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 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 environments
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.
[0003] 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.
[0004] Although such early tests are available, even such tests are
not without flaws. For example, diagnosis of a heart attack within
six hours of the onset of chest pain is difficult to perform with a
single test. While the cardiac marker 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
diagnosis.
[0005] The foregoing example of heart attack is merely one example
of a myriad of diagnoses, which if to be carried out with a high
degree of accuracy, need additional, corroborative tests. Although
the combination of multiple assays performed simultaneously
increases diagnostic 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.
[0006] 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 INVENTION
[0007] In its broadest aspect, the present invention provides an
assay useful to determine the relative levels at which different
analytes are present in a sample. In the present assay, the extent
of 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.
[0008] 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 the result. The method of the invention
simplifies decision making by internally integrating the results of
at least two individual analyte levels.
[0009] 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 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 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 marker) 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.
[0010] 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.
[0011] 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
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
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 first analyte is labeled, and this is ultimately
reflected in the readout obtained following performance of the
assay.
[0012] Thus, in one of its aspects, the present invention provides
a method useful to assay a sample 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.
[0013] 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.
[0014] 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
[0015] (a) forming a reaction mixture by contacting the sample with
reagent means for labeling the first analyte, the labeling reagent
means comprising a mobile, labeled binding partner to the second
analyte, and a conjugate between the second analyte and a binding
partner to the first analyte;
[0016] (b) contacting the reaction mixture with an immobilized
binding partner to the first analyte;
[0017] 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 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 second analyte
in the specimen to become bound thereto, and then presenting the
conjugate 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
allowing it to react first with the labeled second analyte binding
partner before allowing the conjugate to compete therewith for
binding.
[0018] 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 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-carbonic anhydrase HI
antibody, the 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.
[0019] 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 desired activities within 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 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.
[0020] In a second embodiment, a homogeneous assay similar to that
above is provided which employs slightly different reagents, but
applies the same principles. In 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
anitibody 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.
[0021] 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
[0022] (a) forming a reaction mixture by contacting the sample with
[0023] (1) a mobile, labeled binding partner to the second analyte,
[0024] (2) a conjugate between the second analyte and streptavidin;
and [0025] (3) a biotinylated binding partner to the first analyte;
and then
[0026] (b) contacting the reaction mixture with an immobilized
binding partner to the first analyte;
[0027] 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 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.
[0028] 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 second
analyte in the sample becomes bound thereto, before addition of the
competitive-binding conjugate between streptavidin and the second
analyte.
[0029] 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 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 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-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.
[0030] 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
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 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 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 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.
[0031] 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 marker 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.
[0032] 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 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 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.
[0033] Thus, in one embodiment, 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 the
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
[0034] (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 [0035] (b)
contacting the resulting sample with an immobilized binding partner
to the other of the marker and the preselected analyte;
[0036] 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.
[0037] In a preferred embodiment, the immobilized binding partner
is a binding partner for the analyte, and the labeled, mobile
binding partner is a binding partner for the marker.
[0038] 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 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 antibody, and the immobilized binding partner to the
preselected analyte may be an anti-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 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.
[0039] In a second embodiment, an assay similar to that above is
provided which employs variations 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 sequential steps of
[0040] (a) first contacting the sample with an analyte labeling
reagent comprising [0041] (1) a mobile, labeled binding partner to
one of the preselected analyte or the marker, [0042] (2) a
conjugate between the marker and one of biotin and streptavidin,
and [0043] (3) a binding partner to the preselected analyte
conjugated to the other of biotin and streptavidin; and then
[0044] (b) contacting the sample with an immobilized binding
partner to the other of the preselected analyte and the marker;
[0045] 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.
[0046] In a preferred embodiment, the analyte labeling reagent
comprises (1) a mobile, labeled binding partner to the marker, (2)
a conjugate between the marker and streptavidin, and (3) a
biotinylated binding partner to the preselected analyte.
[0047] In other preferred embodiments, the immobilized binding
partner is a binding partner for the analyte.
[0048] 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 binding partner to the
preselected analyte may be an immobilized anti-myoglobin monoclonal
antibody.
[0049] The aforementioned conjugate between the marker and
streptavidin may be a covalent conjugate prepared, for example, by
use of a homobifunctional or heterobifunctional cross-linking 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.
[0050] 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 partners 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 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 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.
[0051] 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,
carbohydrate, nucleic acid, lipid, glycoprotein, glycolipid, by way
of example, but it is not so 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 human. In a preferred embodiment, the sample is whole
blood, the preselected analyte is myoglobin, and the marker is
carbonic anhydrase III.
[0052] 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 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.
[0053] 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 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 markers, 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) together with myoglobin. Thus, the first method described
hereinabove employs a test strip 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-carbonic 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 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.
[0054] 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, 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-carbonic 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 available to bind to the immobilized complex.
[0055] A reduced or negative result will be obtained, depending on
the amount of carbonic anhydrase III in the sample.
[0056] 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
likewise embraced herein.
[0057] It will further be appreciated that formats alternative to
the lateral flow, strip-based format 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 convenient 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 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.
[0058] 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 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.
[0059] 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-binding epitope thereof, to provide a reagents useful
in the practice of the invention. For 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
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
linking, with or without a spacer, one functional group of a
biomolecule to another is well known in the art.
[0060] 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
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 reagents may be made by, for example, a
bifunctional cross-linking reagent to covalently 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 immunoglobulin 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.
[0061] 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.
[0062] 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 preferred embodiment, the analyte or fragment thereof is a
protein or peptide. In 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.
[0063] 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. In 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. 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.
[0064] 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:
Kit I
[0065] 1) a labeled binding partner to the analyte;
[0066] 2) a conjugate between the marker and a second binding
partner to the analyte; and
[0067] 3) an immobilized antibody to the marker.
Kit II
[0068] 1) a labeled binding partner to the marker
[0069] 2) a conjugate between the marker and a binding partner to
the analyte; and
[0070] 3) an immobilized second binding partner to the analyte.
Kit III
[0071] 1) a labeled binding partner to the analyte;
[0072] 2) either [0073] a) a second binding partner to the analyte
conjugated to biotin, and [0074] b) a conjugate between the marker
and a biotin-binding reagent or [0075] c) a second binding partner
to the analyte conjugated to a biotin-binding reagent, and [0076]
d) a conjugate between the marker and biotin; and
[0077] 3) an immobilized binding partner to the marker.
Kit IV
[0078] 1) a labeled binding partner to the marker
[0079] 2) either [0080] a) a binding partner to the analyte
conjugated to biotin and [0081] b) a conjugate between the marker
and a biotin-binding reagent or [0082] c) a binding partner to the
analyte conjugated to a biotin-binding reagent, and [0083] d) a
conjugate between the marker and biotin; and
[0084] 3) an immobilized second binding partner to the analyte.
[0085] 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 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
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 included with the foregoing
reagents.
[0086] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE FIGURES
[0087] FIG. 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 capture line, and two
mobile conjugates, representing the analyte labeling reagent, are
utilized: a detectable (gold-labeled) anti-CAIII antibody
conjugate, and a CAIII-anti-myoglobin antibody conjugate.
[0088] FIG. 2A depicts schematically another embodiment as in FIG.
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.
[0089] FIG. 2B depicts schematically another embodiment as in FIG.
2A, but using a heterogeneous format.
[0090] FIG. 3 depicts graphically the myoglobin, carbonic anhydrase
III, and the ratio therebetween, in a series of myocardial
infarction patients.
[0091] FIG. 4 depicts graphically the myoglobin, carbonic anhydrase
III, and the ratio therebetween, in a series of patients with
muscle disease.
[0092] FIG. 5 shows a depiction of an assay of the invention, with
levels of the components used in modeling the signal needed to
achieve the desired readout.
[0093] FIG. 6 shows a theoretical analysis of the signal needed for
various combinations of levels of myoglobin and carbonic
anhydrase.
[0094] FIG. 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.
[0095] FIG. 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.
[0096] FIG. 9 shows the signal generated from a mathematical model
of the present assay from a range of streptavidin-carbonic
anhydrase III conjugate.
[0097] FIG. 10 shows the signal generated from a mathematical model
of the present assay from a range of biotinylated antibody
levels.
[0098] FIG. 11 shows the signal generated from a mathematical model
of the present assay from a range of gold-conjugated antibody.
[0099] FIG. 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 also to an
anti-carbonic anhydrase III antibody.
[0100] FIG. 13 shows the electrophoretic purity of the
streptavidin-carbonic anhydrase III single-chain polypeptide
described in FIG. 12.
DETAILED DESCRIPTION OF THE INVENTION
[0101] 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 unrelated 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.
[0102] "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.
[0103] "Non-target-bodily-source marker," also referred to simply
as "marker" for brevity, refers to an analyte that is co-released
with the preselected analyte into the bodily fluid from source(s)
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 being assessed by the methods
herein.
[0104] "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, 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.
[0105] This application claims priority under 35 U.S.C. .sctn.
119(e) to Provisional Application Ser. Nos. 60/227,536, filed Aug.
24, 2000, and 60/292,497, filed May 21, 2001, both of which are
incorporated by reference herein in their entireties.
[0106] 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 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 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.
[0107] This format may be used in several ways, one of which is to
read out the ratio of differences 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 specific to that second source. This
may be performed qualitatively or quantitatively by the methods
herein.
[0108] 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
heart-muscle-specific marker which, if detected at elevated levels
in circulation, is absolutely diagnostic of a heart attack.
However, it is not present in detectable levels until about 6 hours
following a heart attack, 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 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.
[0109] 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 marker 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 therapy (and lack
of complications) is critically dependent on an accurate
diagnosis.
[0110] 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 and carbonic 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 marker 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 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.
[0111] 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 affinities of the binding
partners, preferably antibodies, of the various reagents of the
assay. Thus, if 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.
[0112] 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 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
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 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.
[0113] In a further example of the methods herein, a method is
provided for determining with an assay the effective filtering
capacity of a filter based on changes in pore size. This is
applicable in vivo to measuring kidney damage, as it is known 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 kidneys 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 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 proportional to the level of
the larger protein, indicating a decline in renal function. Thus, a
simple renal function test in a test strip format may be prepared
following the teachings herein. In a specific embodiment, the
analyte pair useful in the assessment of kidney damage is alkaline
phosphatase, a normally-secreted small protein, and
5'-nucleotidase, a larger protein the appearance of which indicates
tissue damage.
[0114] 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.
TABLE-US-00001 Test Immobilized Labeling Complex Analyte Marker
reagent Capture Conjugate Labeling Reagent Myo CA3 MyoAb.sub.1(1)
MyoAb.sub.2:Bn SAV:CA3 CA3Ab*(2) Myo CA3 MyoAb.sub.1
MyoAb.sub.2:CA3 CA3Ab* Myo CA3 CA3Ab.sub.1 CA3:myoAb.sub.1, or
MyoAb2* CA3:SAV + MyaAb.sub.1:bn Myo FABP MyoAb.sub.1
MyoAb.sub.2:Bn SAV:FABP FABPAb*(3) Myo MLC MyoAb.sub.1
MyoAb.sub.2:Bn SAV:MLC MLCAb*(4) Alk-Phos 5'-NTase
Alk-PhosAb.sub.1(5) Alk- SAV:5'NTase 5'NTaseAb*(6) PhosAb.sub.2:Bn
LDL HDL LDLAb.sub.1 LDLAb.sub.2:Bn SAV:HDL HDLAb*(7) HDL LDL
HDLAb.sub.1 HDLAb.sub.2:Bn SAV:LDL LDLAb*(8) Key to abbreviations:
*detectable label Myo = myoglobin Ab = antibody, with subscripts
denoting antibodies that recognize 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 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 (IgG1), H86142M (IgGI), H86423 (IgG2a), K31013
(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 (IgG1), H86294M (IgG2b),
and from Spectral Diagnostics under cat # MA-6010. (4)MLC Abs can
be obtained from Spectral Diagnostics under cat # 5010 or MLCI-14.
(5)Alk-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 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).
[0115] As mentioned above, these assays and commercial 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.
[0116] To describe the general invention in another manner, let
A.sub.t represent the first analyte in a sample originating from
the target source, and A.sub.n be the same analyte originating from
the non-target source. Let B represent a second analyte which is
released in proportion to A.sub.n only from the non-target source.
The desirable measurement is A.sub.t 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.sub.n is proportional
to the level of B in the sample, the value of A.sub.t can be
obtained using (A.sub.t plus A.sub.n) minus B. The assay measures
A.sub.t plus A.sub.n but reduces the value with increasing levels
of B in the sample.
[0117] 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, picking 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.
[0118] The relative ratio of release of both the analyte and the
co-released marker 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 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.
[0119] 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 analyte 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 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 nontarget-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
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 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.
[0120] 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
multiple-analyte test strip, etc. Microtiter plate-based or
microbead-based assays are also 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.
[0121] 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 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 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.
[0122] Thus, an assay format is preferred, in which binding
partners such as antibodies can be 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, etc. The
particular selection of binding partners is not limiting, provided
that the binding 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.
[0123] 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 and contact an immobilized
reagent at the capture line. Various format are available to
achieve this purpose, which may be selected by the skilled
artisan.
[0124] 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 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.
[0125] The conjugates of the invention may be prepared by
conventional methods, such as by 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, immobilization of antibodies on
membrane surfaces, etc., are all methods known to one of 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 linked 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
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
site(s) as well as epitopes of the marker necessary for the
operation of the herein described assay. Polynucleotides expressing
such polypeptides, microorganisms and eukaryotic cells expressing
and producing such products including antibodies which 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 partners for the preselected
analyte other than an antibody, such as a receptor, may be provided
in a conjugate with the marker 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, as well as considerations such
as ratios between the components, binding affinities, color
density, etc., are embraced herein.
[0126] One example of such a single-chain polypeptide is described
as SEQ ID NO: 1, an expression product of a polynucleotide (SEQ ID
NO: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 NO: 1. The polynucleotide sequences embraced
herein include degenerate variants which encode the same
polypeptide sequences, by virtue of the 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.
[0127] As noted in the Summary above, the assays of the invention
may be provided in two 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, 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 marker 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 marker and biotin. Both orientations and reagent
selections are fully embraced herein and may be selected depending
on the desired sensitivity of the assay, the characteristics of the
reagents, the relative ratios of analyte to marker encountered, and
other parameters.
[0128] 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 kit 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 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 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 than the first
binding partner mentioned, the following kits are non-limiting
examples of those embraced herein:
Kit I
[0129] 1) a labeled binding partner to the analyte;
[0130] 2) a conjugate between the marker and a second binding
partner to the analyte; and
[0131] 3) an immobilized antibody to the marker.
Kit II
[0132] 1) a labeled binding partner to the marker;
[0133] 2) a conjugate between the marker and a binding partner to
the analyte; and
[0134] 3) an immobilized second binding partner to the analyte.
Kit III
[0135] 1) a labeled binding partner to the analyte;
[0136] 2) a second binding partner to the analyte conjugated to
biotin;
[0137] 3) a conjugate between the marker and a biotin-binding
reagent; and
[0138] 4) an immobilized binding partner to the marker.
Kit IV
[0139] 1) a labeled binding partner to the analyte;
[0140] 2) a second binding partner to the analyte conjugated to a
biotin-binding reagent;
[0141] 3) a conjugate between the marker and biotin; and
[0142] 4) an immobilized binding partner to the marker.
Kit V
[0143] 1) a labeled binding partner to the marker;
[0144] 2) a binding partner to the analyte conjugated to
biotin;
[0145] 3) a conjugate between the marker and a biotin-binding
reagent; and
[0146] 4) an immobilized second binding partner to the analyte.
Kit VI
[0147] 1) a labeled binding partner to the marker;
[0148] 2) a binding partner to the analyte conjugated to a
biotin-binding reagent;
[0149] 3) a conjugate between the marker and biotin; and
[0150] 4) an immobilized second binding partner to the analyte.
[0151] Kit VII
[0152] 1) a conjugate between the marker and a binding partner to
the analyte.
Kit VIII
[0153] 1) a second binding partner to the analyte conjugated to
biotin, and
[0154] 2) a conjugate between the marker and a biotin-binding
reagent.
Kit IX
[0155] 1) a binding partner to the analyte conjugated to a
biotin-binding reagent, and
[0156] 2) a conjugate between the marker and biotin.
[0157] 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 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 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 foregoing kits.
[0158] 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 the following reagents in a preferred
embodiment:
Kit I
[0159] 1) a labeled antibody to myoglobin;
[0160] 2) a conjugate between CAIII and a second antibody to
myoglobin; and
[0161] 3) an immobilized antibody to CAIII.
Kit II
[0162] 1) a labeled antibody to CAIII;
[0163] 2) a conjugate between CAIII and an antibody to myoglobin;
and
[0164] 3) an immobilized second antibody to myoglobin.
Kit III
[0165] 1) a labeled antibody to myoglobin;
[0166] 2) a second antibody to myoglobin conjugated to biotin;
[0167] 3) a conjugate between CAIII and streptavidin; and
[0168] 4) an immobilized antibody to CAIII.
Kit IV
[0169] 1) a labeled antibody to myoglobin;
[0170] 2) a second antibody to myoglobin conjugated to
streptavidin;
[0171] 3) a conjugate between the CAIII and biotin; and
[0172] 4) an immobilized antibody to CAIII.
Kit V
[0173] 1) a labeled antibody to CAIII;
[0174] 2) an antibody to myoglobin conjugated to biotin;
[0175] 3) a conjugate between CAIII and streptavidin; and
[0176] 4) an immobilized second antibody to myoglobin.
Kit VI
[0177] 1) a labeled antibody to CAIII;
[0178] 2) an antibody to myoglobin conjugated to streptavidin;
[0179] 3) a conjugate between CAIII and biotin; and
[0180] 4) an immobilized second antibody to myoglobin.
Kit VII
[0181] 1) a conjugate between CAIII and an antibody to
myoglobin.
Kit VIII
[0182] 1) a second antibody to myoglobin conjugated to biotin,
and
[0183] 2) a conjugate between CAIII and streptavidin.
Kit IX
[0184] 1) an antibody to myoglobin conjugated to streptavidin,
and
[0185] 2) a conjugate between CAIII and biotin.
[0186] 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 thereof and a light or heavy immunoglobulin
chain, which is then assembled to provide a hybrid molecule of an
anti-myoglobin antibody and CAIII.
[0187] Moreover, a kit of the invention may comprise a
polynucleotide encoding a single-chain polypeptide comprising CAIII
and streptavidin.
[0188] The foregoing examples of kits for detecting cardiac
myoglobin in a bodily fluid are merely exemplary of this
embodiment, and as mentioned above, the reagents may be tailored to
measure a large number of other analytes and markers for various
purposes.
[0189] 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.
EXAMPLE 1
Two-Conjugate, Qualitative Homogeneous Assay for Heart Attack
[0190] FIG. 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-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. Immobilized 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 application Ser. No. 09/130,164, filed Aug. 6, 1998, now
U.S. Pat. No. 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 and sedentary life style, is applied to the device, and the
results are read after the test 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 anhydrase 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.
[0191] 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 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 antimyoglobin antibody forms are a consequence of
skeletal 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 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.
EXAMPLE 2
Three-Conjugate, Qualitative and Quantitative Assays for Heart
Attack
[0192] FIG. 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 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 U.S. Pat. No. 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 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 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.
[0193] This result is diagnostic of a heart attack. The patient is
treated immediately with fibrinolytic therapy.
[0194] Similarly, and as shown in FIG. 2B, the same assay depicted
in FIG. 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 Coating
Buffer, pH 9.6 (always prepare in glass). Seal the plate with plate
sealer and store at 4 C overnight. 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 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. 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% H.sub.2O.sub.2). 12. Incubate for 30 min at room
temperature in the dark. 13. Stop reaction with 50 microliters/well
of 4 N H.sub.2SO.sub.4 and read at 490 nm in a plate reader.
[0195] The results from 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.
[0196] 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.
[0197] 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 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 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
[0198] 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 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 attack. The assay
system employed is that described in Example 2, above.
[0199] 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 patients were plotted (from highest to lowest
myoglobin level) along with the ratio between the markers (FIGS. 3
and 4, respectively). The myocardial infarct patient mean ratio was
40.6, with a range of 4.6 to 1 70; 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.
[0200] A model system with the necessary reagents was set up,
according to FIG. 5. The detectable signal achieved by the assay at
various levels of carbonic anhydrase III and myoglobin are shown in
FIG. 6, with a horizontal line drawn at a signal level of 4.6 for
heart attack patients (and 5.06 for non-heart attack patients), the
detectable cut-off level. Using a myoglobin:carbonic anhydrase
ratio of 2.9 and a free carbonic anhydrase level of 2246 ng/ml
(8.02.times.10.sup.-8 M), the signal generated over a range of
myoglobin levels is shown in FIG. 7. Likewise, in FIG. 8, the
signal generated over a range of carbonic anhydrase values for a
ratio of 2.9 and a free myoglobin level of 6467 ng/ml
(3.59.times.10.sup.-7 M) is shown.
[0201] FIG. 9 depicts the effect of different amount of the
streptavidin-carbonic anhydrase III (SAVCAIII) conjugate, and FIG.
10 the amount of biotinylated mobile antibody. FIG. 11 shows the
signal for a range of gold-labeled antibody.
EXAMPLE 4
Preparation of a Single-Chain Streptavidin-Carbonic Anhydrase III
Polypeptide (SAV-CAIII)
[0202] 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. 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 FIG. 12A-B. This polynucleotide (SEQ
ID NO:2) was inserted into pet expression vector and expressed in
E. coli.
[0203] The polypeptide retained biotin-binding affinity as well as
recognition by anti-carbonic anhydrase III antibodies. It was
electrophoretically pure (FIG. 13). A test utilizing a gold-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 BIA core 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.
EXAMPLE 5
Assay of the Invention
[0204] In a demonstration of the operation of the assay of the
invention, the following reagents were used. A rabbit
anti-myoglobin capture antibody was immobilized to a surface. The
mobile reagents included 1) a biotinylated anti-myoglobin antibody
(2 MB-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.
[0205] 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 skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
[0206] Various publications are cited herein, the disclosures of
which are incorporated herein by reference in their entireties.
Sequence CWU 1
1
21421PRTArtificial Sequencesingle-chain polypeptide comprising
carbonic anhydrase III and streptavidin 1Met Asp Pro Ser Lys Asp
Ser Lys Ala Gln Val Ser Ala Ala Glu Ala1 5 10 15Gly Ile Thr Gly Thr
Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val 20 25 30Thr Ala Gly Ala
Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val 35 40 45Gly Asn Ala
Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala 50 55 60Pro Ala
Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp65 70 75
80Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln
85 90 95Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu
Thr 100 105 110Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu
Val Gly His 115 120 125Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala
Ser Ile Asp Ala Ala 130 135 140Lys Lys Ala Gly Val Asn Asn Gly Asn
Pro Leu Asp Ala Val Gln Gln145 150 155 160Thr Arg Ala Lys Glu Trp
Gly Tyr Ala Ser His Asn Gly Pro Asp His 165 170 175Trp His Glu Leu
Phe Pro Asn Ala Lys Gly Gly Asn Gln Ser Pro Val 180 185 190Glu Leu
His Thr Lys Asp Ile Arg His Asp Pro Ser Leu Gln Pro Trp 195 200
205Ser Val Ser Tyr Asp Gly Gly Ser Ala Lys Thr Ile Leu Asn Asn Gly
210 215 220Lys Thr Cys Arg Val Val Phe Asp Asp Thr Tyr Asp Arg Ser
Met Leu225 230 235 240Arg Gly Gly Pro Leu Pro Gly Pro Tyr Arg Leu
Arg Gln Phe His Leu 245 250 255His Trp Gly Ser Ser Asp Asp His Gly
Ser Gly His Thr Val Asp Gly 260 265 270Val Lys Tyr Ala Ala Glu Leu
His Leu Val His Trp Asn Pro Lys Tyr 275 280 285Asn Thr Phe Lys Glu
Ala Leu Lys Gln Arg Asp Gly Ile Ala Val Ile 290 295 300Gly Ile Phe
Leu Lys Ile Gly His Glu Asn Gly Glu Phe Gln Ile Phe305 310 315
320Leu Asp Ala Leu Asp Lys Ile Lys Thr Lys Gly Lys Glu Ala Pro Phe
325 330 335Thr Lys Phe Asp Pro Ser Cys Leu Phe Pro Ala Cys Arg Asp
Tyr Trp 340 345 350Thr Tyr Gln Gly Ser Phe Thr Thr Pro Pro Cys Glu
Glu Cys Ile Val 355 360 365Trp Leu Leu Leu Lys Glu Pro Met Thr Val
Ser Ser Asp Gln Met Ala 370 375 380Lys Leu Arg Ser Leu Leu Ser Ser
Ala Glu Asn Glu Pro Pro Val Pro385 390 395 400Leu Val Ser Asn Trp
Arg Pro Pro Gln Pro Ile Asn Asn Arg Val Val 405 410 415Arg Ala Ser
Phe Lys 42021266DNAArtificial Sequencepolynucleotide sequence
encoding single-chain polypeptide comprising carbonic anhydrase III
and streptavidin 2atggacccct ccaaggactc gaaggcccag gtctcggccg
ccgaggccgg catcaccggc 60acctggtaca accagctcgg ctcgaccttc atcgtgaccg
cgggcgccga cggcgccctg 120accggaacct acgagtcggc cgtcggcaac
gccgagagcc gctacgtcct gaccggtcgt 180tacgacagcg ccccggccac
cgacggcagc ggcaccgccc tcggttggac ggtggcctgg 240aagaataact
accgcaacgc ccactccgcg accacgtgga gcggccagta cgtcggcggc
300gccgaggcga ggatcaacac ccagtggctg ctgacctccg gcaccaccga
ggccaacgcc 360tggaagtcca cgctggtcgg ccacgacacc ttcaccaagg
tgaagccgtc cgccgcctcc 420atcgacgcgg cgaagaaggc cggcgtcaac
aacggcaacc cgctcgacgc cgttcagcag 480actagggcca aggagtgggg
ctacgccagt cacaacggtc ctgaccactg gcatgaactt 540ttcccaaatg
ccaaggggga aaaccagtcg cccgttgagc tgcatactaa agacatcagg
600catgaccctt ctctgcagcc atggtctgtg tcttatgatg gtggctctgc
caagaccatc 660ctgaataatg ggaagacctg ccgagttgta tttgatgata
cttatgatag gtcaatgctg 720agagggggtc ctctccctgg accctaccga
cttcgccagt ttcatcttca ctggggctct 780tcggatgatc atggctctga
gcacaccgtg gatggagtca agtatgcagc ggagcttcat 840ttggttcact
ggaacccgaa gtataacact tttaaagaag ccctgaagca gcgcgatggg
900atcgctgtga ttggcatttt tctgaagata ggacatgaga atggcgagtt
ccagattttc 960cttgatgcat tggacaagat taagacaaag ggcaaggagg
cgcccttcac aaagtttgac 1020ccatcctgcc tgttcccggc atgccgggac
tactggacct accagggctc attcaccacg 1080ccgccctgcg aggaatgcat
tgtgtggctg ctgctgaagg agcccatgac cgtgagctct 1140gaccagatgg
ccaagctgcg gagcctcctc tccagtgctg agaacgagcc cccagtgcct
1200cttgtgagca actggcgacc tccacagcct atcaataaca gggtggtgag
agcttccttc 1260aaatga 1266
* * * * *