U.S. patent number RE39,816 [Application Number 11/117,772] was granted by the patent office on 2007-09-04 for conjunctive analysis of biological marker expression for predicting cardiac mortality.
This patent grant is currently assigned to Nanogen Inc.. Invention is credited to George Jackowski, Eric B. Stanton.
United States Patent |
RE39,816 |
Stanton , et al. |
September 4, 2007 |
Conjunctive analysis of biological marker expression for predicting
cardiac mortality
Abstract
A diagnostic tool is disclosed for accurately and rapidly
diagnosing the condition of an ailing organ. Although applicable to
numerous organ and organ systems, this application particularly
illustrates the concept of conjunctive marker utilization as it
relates to diagnosing and distinguishing congestive heart failure.
The invention particularly relates to the conjunctive utilization
of cardiac Troponin I (cTn-I) and natriuretic peptide, e.g. ANP,
pro-ANP, BNP, pro-BNP and CNP as a retrospective tool for
diagnosing the underlying mechanism of heart failure and as a
prospective analytical device for monitoring disease progression
and efficacy of therapeutic agents.
Inventors: |
Stanton; Eric B. (Burlington,
CA), Jackowski; George (Kettleby, CA) |
Assignee: |
Nanogen Inc. (San Diego,
CA)
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Family
ID: |
25484044 |
Appl.
No.: |
11/117,772 |
Filed: |
April 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
09946171 |
Sep 4, 2001 |
06461828 |
Oct 8, 2002 |
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Current U.S.
Class: |
435/7.92;
422/413; 422/421; 435/7.93; 435/7.94; 435/969; 435/970; 435/973;
435/975; 436/514; 436/518; 436/528; 436/530; 436/807; 436/808;
436/810 |
Current CPC
Class: |
G01N
33/558 (20130101); G01N 33/6893 (20130101); G01N
2800/325 (20130101); Y10S 435/975 (20130101); Y10S
436/807 (20130101); Y10S 436/808 (20130101); Y10S
435/97 (20130101); Y10S 435/973 (20130101); Y10S
436/81 (20130101); Y10S 435/969 (20130101) |
Current International
Class: |
G01N
33/543 (20060101) |
Field of
Search: |
;435/7.92,7.93,7.94,969,970,973,975,528,530,807,808,810
;436/514,518 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 91/09627 |
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Jul 1991 |
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WO |
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WO 02/083913 |
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Oct 2002 |
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WO |
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WO 03/020123 |
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Mar 2003 |
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WO |
|
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Primary Examiner: Nguyen; Bao-Thuy L.
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
What is claimed is:
.[.1. A method for predicting cardiac mortality rate in a patient,
said method comprising the steps of: drawing a sample of serum or
plasma from a patient, depositing the sample in a sample window of
a diagnostic test kit, said test kit comprising a front panel
comprising a sample window and a display window; a back panel; and
a dry chemistry membrane affixed between the front and back panels
positioned for display in at least the display window, wherein said
membrane comprises: a sample region, and a control region, said
sample region positioned to receive the sample from the sample
window; and at least two antibody pairs located at discrete
locations along said membrane between the sample region and the
control region, each of said antibody pairs comprising an antibody
reagent member and an immobilized capture antibody member, each
capture antibody member being located on said membrane closer to
the control region than the corresponding antibody reagent member,
each antibody pair having a measurable or observable moiety labeled
or chemically bonded to the antibody reagent member of each said
antibody pairs, the antibody pairs being monoclonal or polyclonal
and comprising: a first antibody pair that specifically binds to a
marker of cell injury selected from the group consisting of
Troponin-T, cardiac Troponin-I, MLC-1, MLC-2, Glycogen
Phosphorylase BB, Ca ATPase, Phospholamban, Myosin Heavy Chain,
Actin, Tropomyosin, Calmodulin, Caldosmon Phospholamban phosphatase
Calsequestrin, Ca.sup.++ pumping adenosine triphosphatase,
Ca.sup.++ transport ATPase, Adenylate cyclase, Protein kinase,
Histidine rich calcium binding protein, Protein phosphatase,
Protein phosphatase 2C, High affinity calcium binding protein, Low
density lipoprotein-binding sarcoplasmic reticulum protein,
Ca.sup.++-requiring protease (m-calpain), and Pyruvate
dehydrogenase, and a second antibody pair that specifically bind to
a marker of organ adaptation selected from the group consisting of
ANP, pro-ANP, BNP, pro-BNP and CNP, such that upon adding sample to
the sample window, analytes present in the sample and complementary
to the antibody pairs will migrate toward the control region,
binding to the antibody pair each of said analytes, producing a
color change proportional to each concentration of analyte present,
and visualizing or measuring the moiety and determining cardiac
mortality rate..].
.[.2. The method in accordance with claim 1 wherein: the marker of
cell injury is cardiac Troponin-I; and the marker of organ
adaptation is atrial natriuretic peptide or pro-atrial natriuretic
peptide..].
.[.3. The method of claim 1 wherein: said body fluid is selected
from the group consisting of blood, a blood product, plasma, serum,
or urine..].
.[.4. A method for predicting cardiac mortality rate in a patient,
said method comprising the steps of: drawing a sample of a body
fluid from a patient, contacting said sample with a first antibody
that specifically binds to a marker of cell injury selected from
the group consisting of Troponin-T, cardiac Troponin-I, MLC-1,
MLC-2, Glycogen Phosphorylase BB, Ca ATPase, Phospholamban, Myosin
Heavy Chain, Actin, Tropomyosin, Calmodulin, Caldesmon
Phospholaiban phosphatase Calsequestrin, Ca.sup.++ pumping
adenosine triphosphatase, Ca.sup.++ transport ATPase, Adenylate
cyclase, Protein kinase, Histidine rich calcium binding protein,
Protein phosphatase, Protein phosphatase 2C, High affinity calcium
binding protein, Low density lipoprotein-binding sarcoplasmic
reticulum protein, Ca.sup.++-requiring protease (m-calpain), and
Pyruvate dehydrogenase, contacting said sample with a second
antibody that specifically binds to a marker of organ adaptation
selected from the group consisting of ANP, pro-ANP, BNP, pro-BNP
and CNP, and providing means for determining binding between each
of said respective markers and each of said respective antibodies,
whereby said binding provides a means for determining cardiac
mortality rate..].
.[.5. The method of claim 4 wherein: said body fluid is selected
from the group consisting of blood, a blood product, plasma, serum,
or urine..].
6. .Iadd.A method for determining a relative risk of cardiac
mortality in a chronic congestive heart failure patient, said
method comprising the steps of: (A) drawing a sample of a body
fluid from said patient, (B) assaying for the presence of a cardiac
marker of cell injury in said sample using: a first antibody that
specifically binds to a cardiac marker of cell injury, wherein said
cardiac marker of cell injury is selected from the group consisting
of cardiac Troponin-T, cardiac Troponin-I, MLC-1, and MLC-2; and
(C) assaying for the presence of a marker of organ adaptation in a
body fluid sample drawn from said patient using: a second antibody
that specifically binds to said marker of organ adaptation, wherein
said marker of organ adaptation is selected from the group
consisting of ANP, N-terminal ANP, BNP, N-terminal BNP and CNP,
wherein when both said marker of cell injury and said marker of
organ adaptation are present in said sample at increased levels as
compared to control samples, said patient has a greater risk of
cardiac mortality..Iaddend.
.Iadd.7. The method of claim 6, wherein said body fluid is selected
from the group consisting of blood, a blood product, plasma, serum,
or urine..Iaddend.
.Iadd.8. The method of claim 7, wherein said body fluid is serum or
plasma..Iaddend.
.Iadd.9. The method of claim 7 or 8, wherein: said first antibody
specifically binds to a cardiac marker of cell injury selected from
the group consisting of cardiac Troponin-T and cardiac Troponin-I;
and said second antibody specifically binds to a marker of organ
adaptation selected from the group consisting of BNP and N-terminal
BNP..Iaddend.
.Iadd.10. The method of claim 9, wherein: said first antibody
specifically binds to cardiac Troponin-I and said second antibody
specifically binds to N-terminal BNP..Iaddend.
.Iadd.11. The method of claim 6, 7 or 8, wherein both said assaying
for the presence of a cardiac marker and said assaying for the
presence of a marker of organ adaptation, comprise the steps of:
(a) depositing said sample in a sample window of a diagnostic test
kit, wherein said test kit comprises a front panel comprising the
sample window and a display window; a back panel; and a dry
chemistry membrane affixed between the front and back panels
positioned for display in at least the display window, wherein said
membrane comprises: a sample region positioned to receive the
sample from the sample window, a control region, and at least a
first antibody pair and a second antibody pair located at discrete
locations along said membrane between the sample region and the
control region, each of said antibody pairs comprising an antibody
reagent member and an immobilized capture antibody member, each
capture antibody member being located on said membrane closer to
the control region than the corresponding antibody reagent member,
each antibody reagent member having a measurable or observable
moiety labeled or chemically bonded thereto, wherein the antibody
reagent member of said first antibody pair is said first antibody
that specifically binds a cardiac marker of cell injury, and the
antibody reagent member of said second antibody pair is said second
antibody that specifically binds a marker of organ adaptation, such
that the cardiac marker of cell injury and the marker of organ
adaptation present in the sample will migrate toward the control
region, bind to a cognate antibody pair to form an immobilized
immunocomplex, and produce a color change proportional to the
concentration of each marker in the sample, and (b) visualizing or
measuring the moiety labeled or chemically bonded to said first
antibody and said second antibody so as to detect the presence of
each marker in said sample..Iaddend.
.Iadd.12. The method of claim 9, wherein both said assaying for the
presence of a cardiac marker and said assaying for the presence of
a marker of organ adaptation, comprise the steps of: (a) depositing
said sample in a sample window of a diagnostic test kit, wherein
said test kit comprises a front panel comprising the sample window
and a display window; a back panel; and a dry chemistry membrane
affixed between the front and back panels positioned for display in
at least the display window, wherein said membrane comprises: a
sample region positioned to receive the sample from the sample
window, a control region, and at least a first antibody pair and a
second antibody pair located at discrete locations along said
membrane between the sample region and the control region, each of
said antibody pairs comprising an antibody reagent member and an
immobilized capture antibody member, each capture antibody member
being located on said membrane closer to the control region than
the corresponding antibody reagent member, each antibody reagent
member having a measurable or observable moiety labeled or
chemically bonded thereto, wherein the antibody reagent member of
said first antibody pair is said first antibody that specifically
binds cardiac Troponin-T or cardiac Troponin-I and the antibody
reagent member of said second antibody pair is said second antibody
that specifically binds BNP or N-terminal BNP, such that the
cardiac marker of cell injury and the marker of organ adaptation
present in the sample will migrate toward the control region, bind
to a cognate antibody pair to form an immobilized immunocomplex,
and produce a color change proportional to the concentration of
each marker in the sample, and (b) visualizing or measuring the
moiety labeled or chemically bonded to said first antibody and said
second antibody so as to detect the presence of each marker in said
sample..Iaddend.
.Iadd.13. The method of claim 10, wherein both said assaying for
the presence of a cardiac marker and said assaying for the presence
of a marker of organ adaptation, comprise the steps of: (a)
depositing said sample in a sample window of a diagnostic test kit,
wherein said test kit comprises a front panel comprising the sample
window and a display window; a back panel; and a dry chemistry
membrane affixed between the front and back panels positioned for
display in at least the display window, wherein said membrane
comprises: a sample region positioned to receive the sample from
the sample window, a control region, and at least a first antibody
pair and a second antibody pair located at discrete locations along
said membrane between the sample region and the control region,
each of said antibody pairs comprising an antibody reagent member
and an immobilized capture antibody member, each capture antibody
member being located on said membrane closer to the control region
than the corresponding antibody reagent member, each antibody
reagent member having a measurable or observable moiety labeled or
chemically bonded thereto, wherein the antibody reagent member of
said first antibody pair is said first antibody that specifically
binds cardiac Troponin-I and the antibody reagent member of said
second antibody pair is said second antibody that specifically
binds N-terminal BNP, such that the cardiac marker of cell injury
and the marker of organ adaptation present in the sample will
migrate toward the control region, bind to a cognate antibody pair
to form an immobilized immunocomplex, and produce a color change
proportional to the concentration of each marker in the sample, and
(b) visualizing or measuring the moiety labeled or chemically
bonded to said first antibody and said second antibody so as to
detect the presence of each marker in said sample..Iaddend.
.Iadd.14. The method of claim 10, wherein said first antibody is a
monoclonal antibody and said second antibody is a monoclonal
antibody..Iaddend.
.Iadd.15. The method of claim 10, wherein said first antibody is a
polyclonal antibody and said second antibody is a polyclonal
antibody..Iaddend.
.Iadd.16. The method of claim 6, wherein said increased levels are
two-fold greater than in the control samples..Iaddend.
.Iadd.17. The method of claim 6, wherein steps (B) and (C) are
carried out simultaneously..Iaddend.
.Iadd.18. The method of claim 10, wherein said increased levels are
two-fold greater than in the control samples..Iaddend.
.Iadd.19. The method of claim 10, wherein steps (B) and (C) are
carried out simultaneously..Iaddend.
.Iadd.20. The method of claim 10, wherein said first antibody is a
monoclonal antibody and said second antibody is a polyclonal
antibody..Iaddend.
.Iadd.21. The method of claim 10, wherein said first antibody is a
polyclonal antibody and said second antibody is a monoclonal
antibody..Iaddend.
.Iadd.22. The method of claim 6, wherein said first antibody
specifically binds to cardiac Troponin-I; and said second antibody
specifically binds to a N-terminal ANP..Iaddend.
Description
.Iadd.This is a reissue of U.S. Pat. No. 6,461,828, which issued
from application Ser. No. 09/946,171, filed Sep. 4,
2001..Iaddend.
FIELD OF THE INVENTION
This invention relates to the concept of conjunctive utilization of
biological markers expressed in response to abnormal pressure,
volume change and stress to a particular organ (e.g. N-terminal ANP
(pro-ANP) in heart tissue) along with the expression of biological
markers that are indicative of tissue damage (e.g. cardiac Troponin
I (cTnI), or fibrosis markers for heart tissue) as a diagnostic
tool to accurately and rapidly diagnose the condition of the ailing
organ. Although this concept is applicable to numerous organ and
organ systems, this application will particularly illustrate the
concept of conjunctive marker utilization with respect to the
heart, specifically with respect to congestive heart failure. The
invention particularly relates to the conjunctive utilization of
cardiac Troponin I (cTnI) and natriuretic peptides, e.g. brain
natriuretic peptide (BNP), N-terminai BNP (pro-BNP)), c-type
natriuretic peptide (CNP), atrial natriuretic peptide (ANP), and
N-terminal ANP (pro-ANP) as a retrospective tool for diagnosing the
underlying mechanism of heart failure and as a prospective
analytical device for monitoring disease progression and efficacy
of therapeutic agents.
BACKGROUND OF THE INVENTION
Congestive heart failure (CHF) effects approximately 4.8 million
Americans. About 400,000 new cases are diagnosed annually and the
condition is responsible for approximately 200,000 deaths per year.
These statistics, in conjunction with the approximately;1 million
hospitalizations annually attributable to CHF, result in an annual
expenditure on the order of $10 billion.
CHF represents a condition which occurs when heart function becomes
insufficient to meet the needs of the vital systems and tissues of
the body. The inability of the heart to pump sufficiently is
correlated to the measured ejection fraction, which is the percent
of the blood pumped out during each heartbeat. An ejection fraction
of between 50% and 75% is considered normal. This inability can be
caused by failure of one or more sides of the heart, typically the
left but also the right side; such failure can result from systolic
or diastolic dysfunction, and may be represented by an ejection
fraction of less than 50% and a resultant backup of fluid and
accumulation of fluid in the lungs. Although less common,
right-sided heart failure will result in fluid backup that
manifests in a swelling of the veins and surrounding body tissues,
inadequate tissue perfusion, fatigue and poor exercise tolerance.
In addition, heart failure can result from diastolic dysfunction.
This can result from disorders such as hypertension, valvular
disease, transient ischemia, infiltrative disorders or congenital
conditions such as hypertrophic cardiomyopathy. Although there are
cases of pure diastolic dysfunction from infiltrative disorders
such as amyloidosis or fibrosis, heart failure patients often have
a combination of both systolic and diastolic dysfunction
contributing to CHF.
The underlying reasons for this failure in heart functionality are
varied. Thinning and weakening of the ventricle walls leads to
dilation and a loss of pumping ability (systolic dysfunction).
Alternatively, loss of elasticity results in a stiffening, which
may result in a diminished volume of the heart's chambers and loss
of pumping capacity (diastolic dysfunction) and cardiac output.
Additionally, abnormalities in the functioning of the heart's
valves can lead to insufficient cardiac output, for which the body
attempts to compensate by causing the heart to increase its heart
rate, hypertrophy and/or dilate. The compensation mechanisms
utilized by the body may lead to architectural changes in the form
of remodeling (especially after MI) or adaptation of the heart
muscle, ultimately leading to irrevocable loss of function. Related
causes of cardiac failure may be one or more conditions such as
coronary artery disease, ischemic heart damage, e.g. damage
resulting from a heart attack, uncontrolled hypertension, the
direct and/or indirect effects of diabetes on the heart, valvular
heart disease, cardiomyopathy, autoimmune response, disease and
abuse by external agents such as alcohol, tobacco, anabolic
steroids, and the like.
Historically, the preliminary diagnosis of CHF requires a history
and a physical examination during which the condition is often
characterized by various signs and symptoms of intra-vascular and
interstitial volume overload, including shortness of breath,
irregular heart rate, abnormal heart rate and signs of edema. To
determine the severity and prognosis of CHF and to more clearly
characterize a particular patient's condition, further diagnostic
tests are usually needed.
Tests which further illustrate the mechanical condition of the
heart are often useful, such tests include, but are not limited to,
exercise stress testing, electrocardiogram,
radionucleidangiography, echocardiography, chest X-ray and
angiography. Echocardiography is presently considered an important
diagnostic test for CHF. By using ultrasound to provide real time
imagery of the beating heart, valve function and blood flow through
the heart can be readily ascertained. Systolic function and
diastolic function or some combination thereof is determinable
through echocardiography.
Laboratory tests including but not limited to blood and urine
testing are often helpful. These may indicate abnormalities in
liver function, kidney function, cholesterol levels; blood sugar
levels, hemoglobin levels, thyroid hormone levels and ANP, BNP, pro
ANP, pro-BNP.
The diagnostic methods for diagnosing and distinguishing CHF, as
outlined above, require numerous steps, expensive technology, and
trained personnel for their performance and subsequent analysis.
Patients may also be exposed to risk of radiation from nuclear
studies or invasive procedures, i.e. heart catheterization. If a
method and device could be provided for distinguishing and
diagnosing CHF via a simplified body fluid test, the results of
which could be interpreted by a lay person, a long felt need would
be satisfied.
DESCRIPTION OF THE PRIOR ART
It is well documented in the literature that several peptides exist
in the atrium of the human heart which possess the ability to
regulate normal extra-cellular fluid parameters including volume
and pressure of liquid in blood vessels. These peptides are
referred to as Atrial Natriuretic Peptides (ANP). Brain natriuretic
peptide or BNP is a peptide isolated initially from pig brain and
hence the name BNP (Sudoh et al., Nature, 332:78-81, 1988). In
humans, this peptide is synthesized by the brain and myocardial
cells and circulates in the bloodstream exerting profound
influences on the heart and kidneys. BNP is structurally related to
the ANP family of peptides and is present in significantly lower
quantities in circulation. The appearance of pro-BNP has been
correlated with the progression of heart failure. However, the
active molecule is BNP which has been found to be beneficial to the
failing heart. It is conceivable that the damage to heart muscle
may result in an inefficient processing of the inactive pro-BNP to
active BNP (which accounts for the observed increase in pro-BNP).
However, due to the inability of the cardiac tissues to process the
pro-BNP to BNP, there is no beneficial effect unless the active
molecule (BNP) is administered externally.
In addition to changes in pro-BNP/BNP during heart failure, an
increase in cardiac Troponin I correlates well with cardiac tissue
damage and appears to be a good predictor of death due to cardiac
failure. During cardiac cell damage and death, cellular contents
are released into the blood stream. Cardiac Troponin I has been
shown to be a specific diagnostic marker of cardiac cell damage
(Circulation 83, 902-912(1991); Clin. Chem. 40, 1291-1295(1994);
Clin. Chem. 41, 312-317 (1995)).
U.S. Pat. No. 6,162,902 entitled "Human BNP-Specific Antibodies"
provides reagents and assays for the quantification of hBNP in
biological fluid samples such as plasma or serum.
U.S. Pat. No. 5,795,725 entitled "Methods for the Assay of Troponin
I and T and Selection of Autoantibodies for use in Immunoassays"
discloses assays and antibodies for detection and quantitation of
cardiac specific Troponin I and Troponin T in body fluids as an
indicator of myocardial infarction.
The present inventor has previously obtained U.S. Pat. Nos.
5,747,274 and 5,604,105, entitled "Method and Device for Diagnosing
and Distinguishing Chest Pain in Early Onset Thereof", the contents
of which is hereby incorporated by reference. The '274 patent
teaches a diagnostic test, and a device for conducting the test,
for assessing whether patient chest pain is cardiac in origin and
for differentiating between unstable angina and myocardial
infarction as a cause of patient chest pain. The diagnostic test
comprises simultaneously detecting at least three selected cardiac
markers with the use of at least three different monoclonal or
polycional antibody pairs, each member of which is complementary to
a different marker, which is released by heart muscle at varying
stages after the onset of chest pain and is indicative of the cause
of the chest pain. Troponin-I is disclosed as a cardiac specific
ischemic marker.
Additionally, U.S. Pat. No. 5,290,678 to Jackowski entitled
"Diagnostic Kit for Diagnosing and Distinguishing Chest Pain in
Early Onset Thereof", the contents of which is further incorporated
by reference herein, discloses a diagnostic test kit for assessing
whether patient chest pain is cardiac in origin and for
differentiating between unstable angina and myocardial infarction
at early onset of patient chest pain. The test kit comprises a
receptacle for receiving and retaining a sample of blood or serum
of the patient and at least three monoclonal or polyclonal
antibodies suspended on a carrier. Each antibody is complementary
to a different protein released by the heart muscle during early
stages of a myocardial infarction and has corresponding reagents
which are independently responsive to each antibody reacting the
complementary protein. The combined response of reagents indicates
the diagnostic condition of the patient.
The prior art fails to teach or suggest the combined use of a cell
injury marker, e.g. cardiac Troponin-I and a marker related to
volume or pressure overload, e.g. an adaptation marker such as a
natriuretic peptide, e.g. pro-ANP, to provide a testing device for
predicting and/or distinguishing congestive heart failure, nor does
it suggest that the combination of these biological markers could
provide both a retrospective tool for diagnosing the underlying
mechanism of heart failure and a prospective analytical device for
monitoring disease progression and efficacy of therapeutic
agents.
SUMMARY OF THE INVENTION
The present invention reduces to practice the concept of
conjunctive utilization of markers that indicate pressure, volume
change and stress to a particular organ (e.g. pro-ANP in heart
tissue) along with markers that are indicative of tissue damage
(e.g. cardiac Troponin I for heart tissue) as well as markers of
fibrosis, as a diagnostic tool to accurately and rapidly diagnose
the condition of the ailing organ. Although this concept is
applicable to numerous organ and organ systems, this application
will illustrate the concept of conjunctive marker utilization with
respect to the heart.
Cardiac Troponin I and BNP (pro-BNP) have previously been utilized
as markers indicative of cardiac tissue damage and pressure, volume
overload and stress to the heart, respectively. However, neither
these molecules nor any other natriuretic peptide, e.g. pro-ANP,
have been used in conjunction as a diagnostic tool to accurately
and rapidly diagnose the condition of the ailing heart.
The instant invention provides the scientific basis for the
development of an immunological test that has the potential to 1)
replace expensive and time-consuming imaging techniques so that the
appropriate therapeutic intervention may be afforded to the patient
soon after arrival into the emergency room, and 2) provide a
simplified means for diagnosing, distinguishing and quantifying
chronic heart failure and the treatment thereof.
While the examples presented herein are for the heart, the
innovative concept of utilizing biochemical markers to distinguish
tissue damage from adaptive mechanisms is applicable to almost any
organ including, but not limited to, the brain, kidneys, the
adrenal glands, pancreas, lungs, eyes and the liver.
In accordance with this invention the term "cell injury" is defined
as including any transient impairment of cell function, and/or cell
death or necrosis as a result of insult or apoptosis.
In accordance with this invention, the term "organ adaptation"
refers to changes in the organ as a result of or in response to
pressure or volume overload stretch, hypertrophy, wall stress, and
the like physiological factors which stress the organ.
A remodeling including myocardial fibrosis (increased cTnI) or
adaptation (increased natriuretic peptide) of the heart muscle may
accompany progression in CHF. Currently, all these changes to the
heart are evident only with the use of expensive heart imaging
techniques.
Accordingly, it is an objective of the instant invention to provide
an analytical test, either via a central laboratory, or
point-of-care test, e.g. a rapid format test, performed on a
biological fluid for diagnosing congestive heart failure, the
result of said test being readily ascertainable without special
training.
It is a further objective of the instant invention to provide a
test capable of ruling out high risk patients with congestive heart
failure, and thereby permitting the most efficient use of medical
resources.
It is yet another objective of the instant invention to describe a
test which exhibits improved diagnostic accuracy over clinical
evaluation.
It is a still further objective of the invention to provide a test
for detecting pre-clinical disease.
It is yet an additional objective of the instant invention to
provide a test which confirms cardiac etiology of symptoms, reduces
the need for cardiac imaging, yields data for determining long term
management and monitoring, and serves as a predictor of
mortality.
It is yet a further objective of the instant invention to provide a
testing device useful in targeting titration of therapies, e.g.
utilization of ACE inhibitors vasodilators, diuretics and the like;
said test being indicative of the prognostic efficacy of said
therapies.
Other objects and advantages of this invention will become apparent
from the following description taken in conjunction with the
accompanying drawings wherein are set forth, by way of illustration
and example, certain embodiments of this invention.
DETAILED DESCRIPTION OF THE INVENTION
It has been known for many years that during a cardiac event, heart
tissue releases certain molecules, typically protein molecules
which are characteristic of the event. Certain of them are released
as a result of both UA and MI, others are released as a result of
MI. It has been suggested that these markers, often called
analytes, be employed in antigen/antibody reactions to recognize
the cause of a cardiac event.
Sensitivity and Specificity
"Sensitivity" as used herein refers to the ability of an antibody
to recognize and react with its analyte antigen when the analyte is
present at very low concentration in a mixture, i.e., blood, serum,
plasma or other blood preparation when that mixture contains
relatively large numbers of other components. Sensitivity in
antigen/antibody reactions is achieved principally by using
antibodies with high affinity for their antigens.
"Specificity" as used herein refers (a) to the specificity of an
antibody for an analyte, i.e., there is no, or minimal, cross
reaction of the antibody with other materials in the sample under
test; and (b) to the specificity of the source of the antibody,
i.e., did it originate in heart tissue or some other tissue and
therefore facilitate diagnosis.
These different types of sensitivity will be referred to herein as
"cell injury sensitivity," i.e., the antibodies recognize cell
injury markers and "organ adaptation sensitivity," i.e., the
antibodies originate from a specific tissue and therefore permit a
correct and prompt diagnosis. In other words, they are tissue
specific. If they originate only from heart tissue, they are
cardiac specific.
Many markers are known to which antibodies, either monoclonal or
polyclonal, have been produced or can be produced by procedures
well known to the skilled artisan. Many of them are not tissue
specific. They originate not only in heart tissue but also in
muscle or other body tissue. Their tissue sensitivity is not
cardiac sensitivity.
The tests according to the invention can be performed at the point
of care by medically trained personnel. For example, emergency
medical service workers can perform a test of the invention at the
site of a medical emergency or in the ambulance on the way to the
hospital. Similarly, medical personal in the emergency room,
cardiac care facility or other point of care location at a hospital
can perform a test of the invention themselves. Naturally and where
clinically appropriate, the patient sample such as blood or any
blood product, plasma, or serum, or urine may be provided to a
hospital laboratory to perform the test.
The invention extends to test materials including reagents in a kit
form for the practice of the inventive method. The materials
comprise the binding partners that are specific to the markers
under detection, and in one embodiment, comprise the antibody or
antibodies, each of which is specific for one of each of the
markers, the presence of which is to be determined.
In an illustrative embodiment, one antibody of each pair specific
for a particular marker is irreversibly immobilized onto a solid
support; this antibody is alternately referred to hereinafter as a
capture antibody. The other antibody specific for the same marker
is labeled, and is capable of moving with a sample to the location
on the solid support of the capture antibody. This antibody is
sometimes referred to herein as the detection antibody.
The present invention correspondingly extends to devices for
conducting the assays, i.e., a device for early determination of
the presence of congestive heart failure. According to one aspect
of this embodiment of the invention there is provided a device
comprising a housing means containing a membrane unit or section,
with a detector section and a capture section, preferably with a
filter section. The detector section contains at least one detector
antibodies specific to an epitope on each of the markers to be
tested for in a patient's sample of blood, serum or plasma. The
capture section contains at least one capture antibodies specific
to another epitope of each of the markers to be detected. The
capture section is positioned distal to the position of the
detector section, wherein the capture antibodies are irreversibly
immobilized in the capture section, the detector antibodies are
reversibly immobilized in the detector section and migrate with the
sample into the capture section, when the device is in use. The
detector antibodies may be suitably labeled to give a measurable
reaction when the marker is present and is bound in accordance with
the process of this invention.
Binding of the binding partner or antibody to its cognate antigen,
the marker, in a sample can be detected by other detection means,
such as optical detection, biosensors, homogenous immunoassay
formats, and the like. Particular optical sensing systems and
corresponding devices are contemplated and are discussed in U.S.
Pat. 5,290,678.
As used herein, the term "marker" refers to a protein or other
molecule that is released from an organ during a cell injury event
or an organ adaptation event. Such markers include, but are not
limited to, proteins or isoforms of proteins that are either unique
to the heart muscle, and/or proteins or isoforms thereof that are
found in tissues other than heart muscle.
The markers of the present invention are released into the blood.
Thus, the invention contemplates assessing the level of the markers
in blood, or any blood product that contains them such as, but not
limited to, plasma, serum, cytolyzed blood (e.g., by treatment with
hypotonic buffer or detergents; see, e.g., International Patent
Publication No. WO 92/08981, published May 29, 1992), and dilutions
and preparations thereof.
The term "above normal" or "above threshold" are used herein to
refer to a level of a marker that is greater than the level of the
marker observed in normal individuals. For some markers, no or
infinitesimally low levels of the markers may be present. For other
markers, detectable levels may be present normally in blood. Thus,
the terms further contemplate a level that is significantly above
the level found in patients. The term "significantly" refers to
statistical significance, and generally means at least a two-fold
greater level of the marker is present. However, a significant
difference between levels of markers depends on the sensitivity of
the assay employed, and must be taken into account for each marker
assay.
The markers which can be used according to the present invention
are any molecules, typically proteins that pass out from the
organ's cells as the cells become damaged or as adaptation occurs.
These proteins can be either in the native form or can be
immunologically detectable fragments of the protein, resulting, for
example, from photolytic digestion of the protein. When the terms
"marker" or "analyte" are used, they are intended to include
fragments thereof that can be immunologically detected. By
"immunologically detectable" is meant that the protein fragments
contain an epitope that is specifically recognized by a cognate
antibody. Examples of cell injury/necrosis markers are listed below
in Table 1.
TABLE-US-00001 TABLE 1 Troponin-T Troponin-I MLC-1 MLC-2 Glycogen
Phosphorylase BB Ca ATPase Phospholamben Myosin Heavy Chain Actin
Tropomyosin Calmodulin Caldermon Phospholamben phosphate
Calsequestrin Ca.sup.++ pumping adenosine triphosphatase Ca.sup.++
transport ATPase Adenylate cyclase Protein kinase Histidine rich
calcium binding protein Protein phosphatase Protein phosphatase 2C
High affinity calcium binding protein Low density
lipoprotein-binding sarcoplasmic reticulum protein
Ca.sup.++-requiring protease (m-calpsin) Pyruvate
dehydrogenease
EXAMPLE
Cardiac troponin I (cTnI) has been validated as a sensitive and
specific marker of myocyte damage and as a predictor of adverse
events in acute coronary syndromes cTnI has been also reported to
be elevated in patients with congestive heart failure. Similarly,
pro-ANP has been reported to be elevated in patients with CHF.
Congestive heart failure is characterized by hemodynamic and
neurohumoral responses to injury that result in progressive cardiac
remodeling, fibrosis and apoptosis. However in patients with
chronic heart failure, it is unclear whether there is a
relationship between either elevated levels of cTnI alone, or in
conjunction with elevated levels of pro-ANP, and survival. We thus
assessed whether detectable levels of cTnI was associated with
survival in 221 chronic heart failure patients. In addition, we
assessed whether cTnI levels in conjunction with pro-ANP levels was
more predictive of survival than each marker individually. These
patients were categorized as Class III or Class IV by NYHA
standards. Criteria for inclusion in the study were: symptomatic
heart failure of New York Heart Association (NYHA) class III and
IV; left ventricular ejection fraction .ltoreq.35% by radionuclide
ventriculography or echocardiography; treatment with digitalis,
diuretics, and angiotensin-converting enzyme inhibitors.gtoreq.60
days; and informed consent. Criteria for exclusion were restrictive
cardiomyopathy, primary valvular heart disease, consideration for
heart transplantation, history of acute myocardial infarction,
coronary artery bypass graft surgery or other cardiac surgery
.ltoreq.60 days, symptom-limiting unstable angina or angina attacks
.gtoreq.3 per week, and history of symptomatic ventricular
arrhythmias. Additional exclusion criteria were ongoing type I
anti-arrhythmic therapy, concomitant use of calcium-channel
blockers or Hydralazine, use of inhaled .beta.-agonists.gtoreq.once
per week, use of oral or intravenous non-digitalis inotropes more
than one week before the baseline assessment, severe pulmonary or
other systemic diseases, hepatic enzymes more than 2 times the
upper limit of normal, and a serum creatinine .gtoreq.270.mu.mol/L.
Conjunctive analysis of cTnI and pro-ANP yielded the following
results.
TABLE-US-00002 Congestive Heart Failure.sup.1: Cardiac Mortality
Rates Based Upon ANP.sup.2 and cTnI.sup.3 Levels *ANP +ve cTnI +ve
**ANP -ve/cTnI -ve Deceased 18 (29%) 10 (38%) 18 (13%) Alive 44
(71%) 16 (62%) 124 (87%) Statistics 1. ANP +ve vs. ANP -ve/cTnI -ve
Pearson chi-square = 7.944 p = 0.0048 RR = 1.91 (CI 1.262-2.887) 2.
cTnI +ve vs. ANP -ve/cTnI -ve Pearson chi-square = 10.52 p = 0.0011
RR = 3.13 (CI 1.586-6.1558) 3. cTnI +ve vs. ANP +ve Pearson
chi-square = 1.818 p = 0.1775 *ANP +ve/ **ANP -ve/ ANP +ve/ ANP
-ve/ cTnI +ve cTnI -ve cTnI -ve cTnI +ve Deceased 5 (55%) 18 (13%)
13 (24%) 5 (29%) Alive 4 (45%) 124 (87%) 40 (76%) 12 (71%)
Statistics 1. ANP +ve/cTnI +ve vs. ANP -ve/cTnI -ve Pearson
chi-square = 12.052 p = 0.0005 RR = 6.96 (CI 2.018-23.983) 2. ANP
+ve/cTnI +ve vs. ANP +ve/cTnI -ve Pearson chi-square = 3.59 p =
0.0579 RR = 3.06 (CI 0.925-10.094) 3. ANP +ve/cTnI +ve vs. ANP
-ve/cTnI +ve Pearson chi-square = 1.699 p = 0.1923 4. ANP +ve/cTnI
-ve vs. ANP -ve/cTnI -ve Pearson chi-square = 4.055 p = 0.0440 RR =
1.72 (CI 1.0489-2.818) 5. ANP -ve/cTnI +ve vs. ANP -ve/cTnI
-Pearson chi-square = 3.437 p = 0.0637 RR = 2.46 (CI 0.9881-6.3393)
*ANP .gtoreq. 3000 pmol/L cTnI +ve - .gtoreq.0.1 ug/L **ANP <
3000 pmol/L cTnI +ve - <0.1 ug/L .sup.1Class III and IV Heart
Failure .sup.2N-terminal Atrial Natriuretic Peptide .sup.3Cardiac
Troponin I
We have performed a simple linear regression analysis of cTnI
levels on pro-ANP levels. We found that the R2 value for two
separate experiments and their combination was 0.0002. F ration for
the model was 0.06. The probability associated with the model was
0.80. Thus we found no evidence that cTnI levels were significantly
dependent on pro-ANP levels.
cTnI and pro-ANP levels are therefore deemed to be independent of
each other in predicting mortality rates as per the linear
regression analysis, and it is concluded that more prognostic
information related to CHF can be garnered by looking at the
markers conjunctively as opposed to individually.
As used herein, the term antibody includes polyclonal and
monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or
an antigen-binding portion thereof, including but not limited to
F(ab) and Fv fragments, single chain antibodies, chimeric
antibodies, humanized antibodies, and a Fab expression library.
Antibodies useful as detector and capture antibodies in the present
invention, may be prepared by standard techniques well known in the
art. The antibodies can be used in any type of immunoassay. This
includes both the two-site sandwich assay and the single site
immunoassay of the non-competitive type, as well as in traditional
competitive binding assays.
Particularly preferred, for ease and simplicity of detection, and
its quantitative nature, is the sandwich or double antibody assay
of which a number of variations exist, all of which are
contemplated by the present invention. For example, in a typical
sandwich assay, unlabeled antibody is immobilized on a solid phase,
e.g. microtiter plate, and the sample to be tested is added. After
a certain period of incubation to allow formation of an
antibody-antigen complex, a second antibody, labeled with a
reporter molecule capable of inducing a detectable signal, is added
and incubation is continued to allow sufficient time for binding
with the antigen at a different site, resulting with a formation of
a complex of antibody-antigen-labeled antibody. The presence of the
antigen is determined by observation of a signal which may be
quantitated by comparison with control samples containing known
amounts of antigen.
The assays may be competitive assays, sandwich assays, and the
label may be selected from the group of well-known labels such as
radioimmunoassay, fluorescent or chemiluminescence immunoassay, or
immunoPCR technology. Extensive discussion of the known immunoassay
techniques is not required here since these are known to those of
skilled in the art. See Takahashi et al. (Clin Chem
1999;45(8):1307) for S100B assay.
Although not wishing to be limited to any particular embodiment,
the panel format exemplified herein is known and is commercially
available. The panel format is similar to a format currently being
used in association with pregnancy testing and is commercially
available under the trade-mark BIOSIGN.
All patents and publications mentioned in this specification are
indicative of the levels of those skilled in the art to which the
invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
It is to be understood that while a certain form of the invention
is illustrated, it is not to be limited to the specific form or
arrangement herein described and shown. It will be apparent to
those skilled in the art that various changes may be made without
departing from the scope of the invention and the invention is not
to be considered limited to what is shown and described in the
specification.
One skilled in the art will readily appreciate that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned, as well as those inherent
therein. The various biomolecules, e.g. antibodies, markers,
oligonucleotides, peptides, polypeptides, biologically related
compounds, methods, procedures and techniques described herein are
presently representative of the preferred embodiments, are intended
to be exemplary and are not intended as limitations on the scope.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention and
are defined by the scope of the appended claims. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the art are
intended to be within the scope of the following claims.
* * * * *