U.S. patent application number 09/906560 was filed with the patent office on 2001-12-06 for detection and determination of the stages of coronary artery disease.
This patent application is currently assigned to Leuven Research & Development VZW. Invention is credited to Collen, Desire J., Holvoet, Paul N..
Application Number | 20010049112 09/906560 |
Document ID | / |
Family ID | 22524551 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049112 |
Kind Code |
A1 |
Holvoet, Paul N. ; et
al. |
December 6, 2001 |
Detection and determination of the stages of coronary artery
disease
Abstract
A method having clinically sufficient degree of diagnostic
accuracy for detecting the presence of coronary artery disease in a
human patient from the general population and for distinguishing
between the stages of the disease in that patient is disclosed. The
stages are, first, the non-acute stage, which is either
asymptomatic coronary artery disease or stable angina, second, the
acute stage known as unstable angina, and, third, the acute stage
known as acute myocardial infarction. The diseased state (as
opposed to the non-diseased state) is indicated by the clinically
significant presence of a first marker in a sample from the
patient. The presence of one of the two acute stages, unstable
angina or acute myocardial infarction, is indicated by the
clinically significant presence of a second marker in a sample from
the patient. The presence of the more severe acute stage known as
acute myocardial infarction is indicated by the clinically
significant presence of a third marker in a sample from the
patient. Preferably the first marker comprises OxLDL, the second
marker comprises MDA-modified LDL, and the third marker is a
troponin. Preferably the OxLDL and MDA-modified LDL are detected
using monoclonal antibodies that can detect the presence of those
markers in undiluted human plasma at concentrations as low as 0.02
milligrams/deciliter.
Inventors: |
Holvoet, Paul N.;
(Kessel-Lo, BE) ; Collen, Desire J.; (London,
GB) |
Correspondence
Address: |
Stephen P. Gilbert, Esq.
BRYAN CAVE LLP
245 Park Avenue
New York
NY
10167-0034
US
|
Assignee: |
Leuven Research & Development
VZW
|
Family ID: |
22524551 |
Appl. No.: |
09/906560 |
Filed: |
July 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09906560 |
Jul 16, 2001 |
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09148158 |
Sep 4, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/7.92 |
Current CPC
Class: |
Y10S 435/973 20130101;
Y10S 436/819 20130101; G01N 2800/32 20130101; Y10S 436/809
20130101; Y10T 436/104165 20150115; Y10S 435/967 20130101; G01N
2800/324 20130101; G01N 2800/323 20130101; G01N 33/6893 20130101;
G01N 33/721 20130101; Y10T 436/105831 20150115; G01N 33/92
20130101 |
Class at
Publication: |
435/7.1 ;
435/7.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543 |
Claims
We claim:
1. A method having a clinically sufficient degree of diagnostic
accuracy for detecting the presence of and for distinguishing
between or among the non-acute and the acute stages of coronary
artery disease for a human patient from the general population, the
non-acute stage of coronary artery disease being either
asymptomatic coronary artery disease or stable angina and the acute
stages of coronary artery disease being unstable angina and acute
myocardial infarction, the method comprising performing step (b)
and performing at least one of steps (a) and (c): (a) testing a
sample from the patient for a clinically significant presence of a
first marker whose presence above a predetermined level can
indicate with a very high degree of diagnostic accuracy the
presence of coronary artery disease; (b) testing a sample from the
patient for a clinically significant presence of second marker
whose presence above a predetermined level can indicate with a very
high degree of diagnostic accuracy the presence of an acute stage
of coronary artery disease; and (c) testing a sample from the
patient for a clinically significant presence of a third marker
whose presence above a predetermined level can indicate with a high
degree of diagnostic accuracy the presence of acute myocardial
infarction.
2. The method of claim 1 wherein the first marker is a first
atherogenic protein.
3. The method of claim 2 wherein the first atherogenic protein
comprises OxLDL containing at least 60 substituted lysine residues
per apo B-100 moiety.
4. The method of claim I wherein the second marker is a second
atherogenic protein.
5. The method of claim 4 wherein the second atherogenic protein
comprises MDA-modified LDL containing at least 60 substituted
lysine residues per apo B-100 moiety.
6. The method of claim 1 wherein the third marker is a heart
protein.
7. The method of claim 6 wherein the third marker is selected from
the group consisting of a troponin and CK-MB.
8. The method of claim 1 wherein step (a) uses an immunological
assay that can indicate the clinically significant presence of the
first marker.
9. The method of claim 8 wherein the immunological assay is a first
sandwich assay.
10. The method of claim 8 wherein the immunological assay uses a
first monoclonal antibody having a high affinity for the first
marker.
11. The method of claim 10 wherein the first monoclonal antibody
has an affinity for the first marker of at least about
1.times.10.sup.10 M.sup.-1.
12. The method of claim 11 wherein the first monoclonal antibody is
mAb-4E6.
13. The method of claim 1 wherein step (b) is conducted using an
immunological assay that can indicate the clinically significant
presence of the second marker.
14. The method of claim 13 wherein the immunological assay is a
second sandwich assay.
15. The method of claim 13 wherein the immunological assay uses a
second monoclonal antibody having a high affinity for the second
marker.
16. The method of claim 15 wherein the second monoclonal antibody
has an affinity for the second marker of at least about
1.times.10.sup.10 M.sup.-1.
17. The method of claim 16 wherein the second monoclonal antibody
is mAb-1H11.
18. The method of claim 1 wherein step (a) is performed.
19. The method of claim 18 wherein the first marker is a first
atherogenic protein comprising OxLDL, and the second marker is a
second atherogenic protein comprising MDA-modified LDL.
20. The method of claim 18 wherein steps (a) and (b) are performed
using immunological assays.
21. The method of claim 20 wherein the assays are performed using
monoclonal antibodies selected from the group consisting of
mAb-4E6, mAb-1H11, and mAb-8A2.
22. The method of claim 20 wherein the immunological assays are
sandwich assays.
23. The method of claim 22 wherein the assays are performed using
monoclonal antibodies selected from the group consisting of
mAb-4E6, mAb-1H11, and mAb-8A2.
24. The method of claim 19 wherein the test of step (a) can detect
the presence of OxLDL containing at least 60 substituted lysine
residues per apo B-100 moiety in undiluted human plasma in a
concentration of 0.02 milligrams/deciliter and the test of step (b)
can detected the presence of MDA-modified LDL containing at least
60 substituted lysine residues per apo B-100 moiety in undiluted
human plasma in a concentration of 0.02 milligrams/deciliter.
25. The method of claim 1 wherein step (c) is performed.
26. The method of claim 25 wherein the third marker is a heart
protein.
27. The method of claim 25 wherein the third marker is selected
from the group consisting of a troponin and CK-MB.
28. The method of claim 1 wherein both of steps (a) and (c) are
performed.
29. The method of claim 28 wherein the first marker is a first
atherogenic protein.
30. The method of claim 29 wherein the first atherogenic protein
comprises OxLDL containing at least 60 substituted lysine residues
per apo B-100 moiety.
31. The method of claim 28 wherein the second marker is a second
atherogenic protein.
32. The method of claim 31 wherein the second atherogenic protein
comprises MDA-modified LDL containing at least 60 substituted
lysine residues per apo B-100 moiety.
33. The method of claim 28 wherein the third marker is a heart
protein.
34. The method of claim 33 wherein the third marker is selected
from the group consisting of a troponin and CK-MB.
35. The method of claim 28 wherein step (a) uses an immunological
assay that can indicate the clinically significant presence of the
first marker.
36. The method of claim 35 wherein the immunological assay is a
first sandwich assay.
37. The method of claim 35 wherein the immunological assay uses a
first monoclonal antibody having a high affinity for the first
marker.
38. The method of claim 37 wherein the first monoclonal antibody
has an affinity for the first marker of at least about
1.times.10.sup.10 M.sup.-1.
39. The method of claim 38 wherein the first monoclonal antibody is
mAb-4E6.
40. The method of claim 28 wherein step (b) is conducted using an
immunological assay that can indicate the clinically significant
presence of the second marker.
41. The method of claim 40 wherein the immunological assay is a
second sandwich assay.
42. The method of claim 40 wherein the immunological assay uses a
second monoclonal antibody having a high affinity for the second
marker.
43. The method of claim 42 wherein the second monoclonal antibody
has an affinity for the second marker of at least about
1.times.10.sup.10 M.sup.-1.
44. The method of claim 43 wherein the second monoclonal antibody
is mAb-1H11.
45. The method of claim 28 wherein the first marker is a first
atherogenic protein comprising OxLDL containing at least 60
substituted lysine residues per apo B-100 moiety and the second
marker is a second atherogenic protein comprising MDA-modified LDL
containing at least 60 substituted lysine residues per apo B-100
moiety.
46. The method of claim 45 wherein steps (a) and (b) are conducted
using immunological assays.
47. The method of claim 46 wherein the immunological assays are
sandwich assays.
48. The method of claim 46 wherein the assays are conducted using
monoclonal antibodies selected from the group consisting of
mAb-4E6, mAb-1H11, and mAb-8A2.
49. The method of claim 45 wherein the test of step (a) can detect
the presence of OxLDL containing at least 60 substituted lysine
residues per apo B-100 moiety in undiluted human plasma in a
concentration of 0.02 milligrams/deciliter and the test of step (b)
can detected the presence of MDA-modified LDL containing at least
60 substituted lysine residues per apo B-100 moiety in undiluted
human plasma in a concentration of 0.02 milligrams/deciliter.
50. The method of claim 49 wherein the third marker is selected
from the group consisting of a troponin and CK-MB.
51. A method having a clinically sufficient degree of diagnostic
accuracy for detecting the presence of and for distinguishing
between or among the non-acute and the acute stages of coronary
artery disease for a human patient from the general population, the
non-acute stage of coronary artery disease being either
asymptomatic coronary artery disease or stable angina and the acute
stages of coronary artery disease being unstable angina and acute
myocardial infarction, the method comprising the steps: (a) testing
a sample from the patient using an immunological assay for a
clinically significant presence of OxLDL containing at least 60
substituted lysine residues per apo B-100 moiety, its presence
above a predetermined level being able to indicate with a very high
degree of diagnostic accuracy the presence of coronary artery
disease, the assay employing at least one monoclonal antibody
having a high affinity for the OxLDL; (b) testing a sample from the
patient using an immunological assay for a clinically significant
presence of MDA-modified LDL containing at least 60 substituted
lysine residues per apo B-100 moiety, its presence above a
predetermined level being able to indicate with a very high degree
of diagnostic accuracy the presence of an acute stage of coronary
artery disease, the assay employing at least one monoclonal
antibody having a high affinity for MDA-modified LDL; and (c)
optionally testing a sample from the patient for a clinically
significant presence of a third marker whose presence above a
predetermined level can indicate with a high degree of diagnostic
accuracy the presence of acute myocardial infarction.
52. The method of claim 51 wherein the assay of step (a) can detect
the presence of the OxLDL in undiluted human plasma in a
concentration of 0.02 milligrams/deciliter and the assay of step
(b) can detected the presence of the MDA-modified LDL in undiluted
human plasma in a concentration of 0.02 milligrams/deciliter.
53. The method of claim 51 wherein the at least one monoclonal
antibody used in the assay of step (a) has an affinity for the
OxLDL of at least about 1.times.10.sup.10 M.sup.-1 and the at least
one monoclonal antibody used in the assay of step (b) has an
affinity for the MDA-modified LDL of at least about
1.times.10.sup.10 M.sup.-1.
54. The method of claim 51 wherein the monoclonal antibodies used
in the assays of steps (a) and (b) are selected from the group
consisting of mAb-4E6, mAb-1H11, and mAb-8A2.
55. The method of claim 51 wherein step (c) is performed and the
third marker is a heart protein.
56. The method of claim 55 wherein the heart protein is selected
from the group consisting of a troponin and CK-MB.
57. A method having a clinically sufficient degree of diagnostic
accuracy for detecting the presence of and for distinguishing
between or among the non-acute and the acute stages of coronary
artery disease for a human patient from the general population, the
non-acute stage of coronary artery disease being either
asymptomatic coronary artery disease or stable angina and the acute
stages of coronary artery disease being unstable angina and acute
myocardial infarction, the method comprising the steps: (a) testing
a sample from the patient using an immunological assay for a
clinically significant presence of OxLDL containing at least 60
substituted lysine residues per apo B-100 moiety, its presence
above a predetermined level being able to indicate with a very high
degree of diagnostic accuracy the presence of coronary artery
disease, the assay employing at least one monoclonal antibody
having a high affinity for the OxLDL; (b) testing a sample from the
patient using an immunological assay for a clinically significant
presence of MDA-modified LDL containing at least 60 substituted
lysine residues per apo B-100 moiety, its presence above a
predetermined level being able to indicate with a very high degree
of diagnostic accuracy the presence of an acute stage of coronary
artery disease, the assay employing at least one monoclonal
antibody having a high affinity for MDA-modified LDL; and (c)
testing a sample from the patient for a clinically significant
presence of a heart protein whose presence above a predetermined
level can indicate with a high degree of diagnostic accuracy the
presence of acute myocardial infarction.
58. The method of claim 57 wherein the monoclonal antibodies used
in the assays of steps (a) and (b) are selected from the group
consisting of mAb-4E6, mAb-1H11, and mAb-8A2 and the heart protein
is selected from the group consisting of a troponin and CK-MB.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of coronary
artery disease. More specifically, it relates to detecting with a
clinically sufficient degree of diagnostic accuracy whether a human
patient from the general population has coronary artery disease
("CAD") and, if so, to determining with a clinically sufficient
degree of diagnostic accuracy which stage of CAD the patient
has.
[0002] Steinberg D, "Lewis A. Conner Memorial Lecture, Oxidative
Modification Of LDL And Atherogenesis," Circulation 1997, 95:
1062-1071, notes that deaths from coronary heart disease continue
to outnumber deaths from any other single cause in the United
States. Kolata, "A New Generation Of Tests To Determine Heart
Trouble," New York Times News Service (Nov. 26, 1995), reports that
half of the 600,000 Americans who have heart attacks each year have
no symptoms beforehand and that as many as 30% of heart disease
patients do not have any obvious risk factors such as high blood
pressure, high cholesterol levels, diabetes, or a family history of
heart disease. (All of the documents mentioned or otherwise
referenced herein are incorporated herein in their entireties for
all purposes.)
[0003] The ability to accurately determine whether a patient has
coronary artery disease and, if so, what stage the patient has, has
been a long-standing (but heretofore unachieved) goal of medical
science. There have been many attempts to provide monoclonal
antibodies that recognize in humans and other animals various low
density lipoprotein ("LDL") substances and/or other substances that
might be associated with atherosclerosis and/or thrombosis. There
have also been attempts to provide methods for determining possible
markers for atherosclerosis and coronary injury. See, e.g., U.S.
Pat. Nos. 5,024,829, 5,026,537, 5,120,834, 5,196,324, 5,223,410,
5,362,649, 5,380,667, 5,396,886, 5,453,359, 5,487,892, 5,597,726,
5,658,729, 5,690,103, and 5,756,067; EPO Published Application 0
484 863 A1; PCT/EP97/03287 (unpublished application);
PCT/EP97/03493 (unpublished application); PCT Published Application
WO 94/23302; Adams et al., "Cardiac Troponin I. A Marker With High
Specificity For Cardiac Injury," Circulation 1993; 88(1): 101-106;
American Biogenetic Sciences Inc., 1995 Annual Report, 24 pages
(1995); American Biogenetic Sciences, Focus on Diagnostic Tests: A
Technology Analysis, Updated Full Report, Paisley and Habermas,
Inc. (Jun. 3, 1996); Antman et al., "Cardiac-Specific Troponin I
Levels To Predict The Risk Of Mortality In Patients With Acute
Coronary Syndromes," N. Eng. J. Med. 1996; 335(18): 1342-1349;
AtheroGenics, Inc. Web Site (WWW.ATHEROGENICS.COM); Hamm et al.,
"Emergency Room Triage Of Patients With Acute Chest Pain By Means
Of Rapid Testing For Cardiac Troponin T Or Troponin I," N. Eng. J.
Med. 1997; 337(23): 1648-1653; Hammer et al., "Generation,
Characterization, And Histochemical Application Of Monoclonal
Antibodies Selectively Recognizing Oxidatively Modified
ApoB-Containing Serum Lipoproteins," Arterioscler. Thromb. Vasc.
Biol. 1995; 15(5): 704-713; Hoff et al., "Lesion-Derived Low
Density Lipoprotein And Oxidized Low Density Lipoprotein Share A
Lability For Aggregation, Leading To Enhanced Macrophage
Degradation," Arterioscler. Thromb. 1991; 11(5): 1209-1222;
Hoffmeister et al., "Alterations Of Coagulation And Fibrinolytic
And Kallikrein-Kinin Systems In The Acute And Post-Acute Phases In
Patients With Unstable Angina Pectoris," Circulation 1995; 91(10):
2520-2527; Holvoet, Collen, et al., "Stimulation With A Monoclonal
Antibody (mAb4E4) Of Scavenger Receptor-Mediated Uptake Of
Chemically Modified Low Density Lipoproteins By THP-1-Derived
Macrophages Enhances Foam Cell Generation," J. Clin. Invest. 1994;
93: 89-98; Holvoet and Collen, ".beta.-VLDL Hypercholesterolemia
Relative To LDL Hypercholesterolemia Is Associated With Higher
Levels Of Oxidized Lipoproteins And A More Rapid Progression Of
Coronary Atherosclerosis In Rabbits," Arterioscler. Thromb. Vasc.
Biol. November 1997; 17(11): 2376-2382; Holvoet and Collen,
"Oxidized Lipoproteins In Atherosclerosis And Thrombosis," FASEB J.
1994; 8: 1279-1284; Holvoet and Collen, "Malondialdehyde-Modified
Low Density Lipoproteins In Patients With Atherosclerotic Disease,"
J. Clin. Invest. 1995; 95: 2611-2619; Holvoet, Collen, et al.,
"Correlation Between Oxidized Low Density Lipoproteins And Von
Willebrand Factor In Chronic Renal Failure," Thromb. Haemost. 1996;
76(5): 663-669; Holvoet, Collen, et al., "Correlation Between
Oxidized Low Density Lipoproteins And Coronary Artery Disease In
Heart Transplant Patients," Abstract published in Final Programme
of 66th Congress of the European Atherosclerosis Society, Florence
(Italy), Jul. 13-14, 1996, Abstract Book, page 47; Holvoet, Collen,
et al., "Oxidized Low Density Lipoproteins In Patients With
Transplant-Associated Coronary Artery Disease," Arterioscler.
Thromb. Vasc. Biol. January 1998; 18(1): 100-107; Holvoet, Collen,
et al., Presentation at 70th Scientific Session Of The American
Heart Association, Orlando, Fla., Nov. 9-12, and published in
abstract form in Circulation 1997; 96(Suppl. I): 1417 (Abstract
2328); Itabe et al., "A Monoclonal Antibody Against Oxidized
Lipoprotein Recognizes Foam Cells In Atherosclerotic Lesions:
Complex Formation Of Oxidized Phosphatidylcholines And
Polypeptides," J. Bio. Chem. 1994; 269(21): 15274-15279; Itabe et
al., "Sensitive Detection Of Oxidatively Modified Low Density
Lipoprotein Using A Monoclonal Antibody," J. Lipid Res. 1996; 37:
45-53; Kolata, "A New Generation Of Tests To Determine Heart
Trouble," New York Times News Service (Nov. 26, 1995); Kotani et
al., "Distribution Of Immunoreactive Malondialdehyde-Modified
Low-Density Lipoprotein In Human Serum," Biochimica et Biophysica
Acta 1994; 1215: 121-125; Menschikowski et al., "Secretory Group II
Phospholipase A2 In Human Atherosclerotic Plaques," Atherosclerosis
1995; 118: 173-181; Muldoon et al., "C-Reactive Protein And Serum
Amyloid A Protein In Unstable Angina," N. Engl. J Med. 1995;
332(6): 398-400; Ohman et al., "Cardiac Troponin T Levels For Risk
Stratification In Acute Myocardial Ischemia," N. Eng. J Med. 1996
335(18): 1333-1341; Palinski et al., "Antisera And Monoclonal
Antibodies Specific For Epitopes Generated During Oxidative
Modification Of Low Density Lipoprotein," Arteriosclerosis 1990;
10(3): 325-335; Ravalli et al., "Immunohistochemical Demonstration
Of 15-Lipoxygenase In Transplant Coronary Artery Disease,"
Arterioscler. Thromb. Vasc. Biol. 1995; 15(3): 340-348; Reade et
al., "Expression Of Apolipoprotein B Epitopes In Low Density
Lipoproteins Of Hemodialyzed Patients," Kidney Int. 1993; 44:
1360-1365; Reverter et al., "Platelet Activation During
Hemodialysis Measured Through Exposure Of P-Selectin: Analysis By
Flow Cytometric And Ultrastructural Techniques," J. Lab. Clin. Med.
1994; 124(1): 79-85; Salonen et al., "Autoantibody Against Oxidised
LDL And Progression Of Carotid Atherosclerosis," Lancet 1992;
339(8798): 883-887; Uchida et al., "Protein-Bound Acrolein:
Potential Markers For Oxidative Stress," Proc. Natl. Acad. Sci. USA
1998; 95: 4882-4887; and Van de Werf, "Cardiac Troponins In Acute
Coronary Syndromes," N. Eng. J Med. 1996; 335(18): 1388-1389.
[0004] However, as noted in the literature, there is no currently
available method for determining with a clinically sufficient
degree of diagnostic accuracy the presence of coronary artery
disease in a patient and, if the disease is present, for
distinguishing with a clinically sufficient degree of diagnostic
accuracy between or among the non-acute (i.e., chronic) and acute
stages of that disease, the non-acute stages being stable angina
and presumably asymptomatic coronary artery disease and the acute
stages being unstable angina and acute myocardial infarction.
[0005] For example, U.S. Pat. No. 5,380,667 (issued Jan. 10, 1995)
notes that most individuals with heart disease are largely
asymptomatic until their first heart attack, that the major risk
factors thus far identified in the prior art are not perfect
predictors (particularly for predicting the risk of coronary artery
disease in any single individual), and that thirty to forty percent
of the population is still misdiagnosed using the known major risk
factors (column 1, lines 31-39).
[0006] Hlatky M A, "Evaluation Of Chest Pain In The Emergency
Department," N. Eng. J. Med. December 1997, 337(23): 1687-1689,
reports that after patients in the emergency department having
clear-cut acute myocardial infarction have been identified, the
remaining patients are more difficult to sort out; that symptoms
suggestive of myocardial ischemia at rest that last more than 15
minutes indicate a relatively high short-term risk, probably
because of their association with ruptured coronary plaque; that
further tests used for patients include those that identify a
defect in myocardial perfusion, abnormalities in left ventricular
wall motion, or subtle evidence of myocardial necrosis though
sensitive assays of intracellular proteins (e.g., CK-MB isoenzyme,
myoglobin, troponin T, and troponin I); that even a highly
sensitive marker of myocardial necrosis will not necessarily be
positive in all patients with acute myocardial ischemia; and that
patients who present for the first time with chest pain usually
need further tests to establish the likelihood of underlying
coronary disease and to guide appropriate therapy.
[0007] U.S. Pat. No. 5,756,067 (issued May 26, 1998) notes that
tests currently available to measure the risk of developing
atherosclerosis include measuring the plasma content of
cholesterol, triglycerides, and lipoproteins but that it is clear
that these tests are not conclusive because approximately one-half
of heart disease due to atherosclerosis occurs in patients with
plasma triglycerides and cholesterol within the normal ranges of
the population and because angiographic evidence of atherosclerosis
has been found in patients with normal lipid levels.
[0008] Sasavage N, "Predicting Coronary Artery Disease, New Markers
Could Identify Patients At Risk," Clin. Lab. News March 1998, pages
6-7, suggests that oxidation of low density lipoproteins may render
it more atherogenic, that detection of oxidized LDL species faces
some technical difficulties, and indicates that coronary artery
disease appears to be a multifactorial disease. It also states that
those who work in this area agree that development of a new
generation of biochemical markers will allow clinicians to better
assess patient risk and intervene with treatments to avoid adverse
outcomes.
[0009] Thus, there is a significant need for a method that with a
clinically sufficient degree of diagnostic accuracy can detect
coronary artery disease and distinguish between or among its
stages.
SUMMARY OF THE INVENTION
[0010] An invention satisfying those needs and having advantages
and benefits that will be apparent to one skilled in the art has
now been developed. Broadly, this invention provides a method
having a clinically sufficient degree of diagnostic accuracy for
detecting the presence of and for distinguishing between or among
the non-acute and the acute stages of coronary artery disease for a
human patient from the general population, the non-acute stage of
coronary artery disease being either asymptomatic coronary artery
disease or stable angina and the acute stages of coronary artery
disease being unstable angina and acute myocardial infarction, the
method comprising performing step (b) and performing at least one
of steps (a) and (c):
[0011] (a) testing a sample from the patient for a clinically
significant presence of a first marker whose presence above a
predetermined level can indicate with a very high degree of
diagnostic accuracy the presence of coronary artery disease;
[0012] (b) testing a sample from the patient for a clinically
significant presence of second marker whose presence above a
predetermined level can indicate with a very high degree of
diagnostic accuracy the presence of an acute stage of coronary
artery disease; and
[0013] (c) testing a sample from the patient for a clinically
significant presence of a third marker whose presence above a
predetermined level can indicate with a high degree of diagnostic
accuracy the presence of acute myocardial infarction.
[0014] In some embodiments of the invention, steps (a) and (b) but
not (c) will be used, in other embodiments steps (b) and (c) but
not (a) will be used, and in still other embodiments all three of
steps (a), (b), and (c) will be used.
[0015] In some embodiments, the first marker is a first atherogenic
protein preferably comprising OxLDL containing at least 60
substituted lysine residues per apo B-100 moiety. In some
embodiments, the second marker is a second atherogenic protein
preferably comprising MDA-modified LDL containing at least 60
substituted lysine residues per apo B-100 moiety. In some
embodiments, the third marker is a heart protein and preferably is
a troponin (e.g., Troponin I) or CK-MB.
[0016] Desirably each of steps (a) and (b) uses an immunological
assay, which is preferably a sandwich assay, although a competitive
assay may be used. Preferably, each immunological assay uses one or
more monoclonal antibody having high affinities for their
respective markers, e.g., affinity of at least about
1.times.10.sup.-1. ("M" indicates molarity or gmoles/liter;
"M.sup.-1" indicates the reciprocal of molarity, or liters per
mole.) The monoclonal antibodies used may be selected from the
group consisting of mAb-4E6, mAb-1H11, and mAb-8A2.
[0017] If the marker of step (a) is OxLDL containing at least 60
substituted lysine residues per apo B-100 moiety, preferably the
test used in step (a) is capable of detecting that substance in
undiluted human plasma in a concentration of 0.02
milligrams/deciliter (0.02 mg/dL). If the marker of step (b) is
MDA-modified LDL containing at least 60 substituted lysine residues
per apo B-1 00 moiety, preferably the test used in step (b) is
capable of detecting that substance in undiluted human plasma in a
concentration of 0.02 milligrams/deciliter (0.02 mg/dL).
[0018] In another aspect, this invention provides a method having a
clinically sufficient degree of diagnostic accuracy for detecting
the presence of and for distinguishing between or among the
non-acute and the acute stages of coronary artery disease for a
human patient from the general population, the non-acute stage of
coronary artery disease being either asymptomatic coronary artery
disease or stable angina and the acute stages of coronary artery
disease being unstable angina and acute myocardial infarction, the
method comprising the steps:
[0019] (a) testing a sample from the patient using an immunological
assay for a clinically significant presence of OxLDL containing at
least 60 substituted lysine residues per apo B-100 moiety, its
presence above a predetermined level being able to indicate with a
very high degree of diagnostic accuracy the presence of coronary
artery disease, the assay employing at least one monoclonal
antibody having a high affinity for the OxLDL;
[0020] (b) testing a sample from the patient using an immunological
assay for a clinically significant presence of MDA-modified LDL
containing at least 60 substituted lysine residues per apo B-100
moiety, its presence above a predetermined level being able to
indicate with a very high degree of diagnostic accuracy the
presence of an acute stage of coronary artery disease, the assay
employing at least one monoclonal antibody having a high affinity
for MDA-modified LDL; and
[0021] (c) optionally testing a sample from the patient for a
clinically significant presence of a third marker whose presence
above a predetermined level can indicate with a high degree of
diagnostic accuracy the presence of acute myocardial
infarction.
[0022] In yet another aspect, this invention provides a method
having a clinically sufficient degree of diagnostic accuracy for
detecting the presence of and for distinguishing between or among
the non-acute and the acute stages of coronary artery disease for a
human patient from the general population, the non-acute stage of
coronary artery disease being either asymptomatic coronary artery
disease or stable angina and the acute stages of coronary artery
disease being unstable angina and acute myocardial infarction, the
method comprising the steps:
[0023] (a) testing a sample from the patient using an immunological
assay for a clinically significant presence of OxLDL containing at
least 60 substituted lysine residues per apo B-1 00 moiety, its
presence above a predetermined level being able to indicate with a
very high degree of diagnostic accuracy the presence of coronary
artery disease, the assay employing at least one monoclonal
antibody having a high affinity for the OxLDL;
[0024] (b) testing a sample from the patient using an immunological
assay for a clinically significant presence of MDA-modified LDL
containing at least 60 substituted lysine residues per apo B-100
moiety, its presence above a predetermined level being able to
indicate with a very high degree of diagnostic accuracy the
presence of an acute stage of coronary artery disease, the assay
employing at least one monoclonal antibody having a high affinity
for MDA-modified LDL; and
[0025] (c) testing a sample from the patient for a clinically
significant presence of a heart protein whose presence above a
predetermined level can indicate with a high degree of diagnostic
accuracy the presence of acute myocardial infarction.
[0026] The clinically significant presence (presence above a
predetermined level) of the first marker (e.g., OxLDL having at
least at least 60 substituted lysine residues per apo B-100 moiety)
can indicate with a very high degree of diagnostic accuracy the
presence of coronary artery disease. In other words, the test or
assay of this invention used for detecting a marker of coronary
artery disease will distinguish with a very high degree of
diagnostic accuracy between the following categories 1 and 2: (1)
those who do not have coronary artery disease and (2) those who do
have one of the categories or stages of coronary artery disease
(i.e., those who have non-acute [or chronic] disease, namely,
stable angina or presumably asymptomatic coronary artery disease,
or those who have acute coronary syndromes clinically presenting as
unstable angina or acute myocardial infarction), but by itself will
generally not be able to distinguish between the categories (or
stages) of coronary artery disease.
[0027] The clinically significant presence (presence above a
predetermined level) of the second marker (e.g., MDA-modified LDL
having at least at least 60 substituted lysine residues per apo
B-100 moiety) can indicate with a very high degree of diagnostic
accuracy the presence of an acute stage of coronary artery disease.
In other words, the test or assay of this invention for detecting a
marker of an acute stage of coronary artery disease will
distinguish between the following categories 1 and 2: (1) those who
do not have an acute stage of coronary artery disease (i.e., those
who have either (a) no coronary artery disease or have non-acute
coronary artery disease, namely, (b) asymptomatic coronary artery
disease or (c) stable angina) but by itself will generally not be
able to distinguish between those three categories a, b, and c, and
(2) those who do have one of the two categories or stages of acute
coronary artery disease (i.e., those who have either (a) unstable
angina or (b) acute myocardial infarction) but by itself will
generally not be able to distinguish between the two acute
categories.
[0028] The clinically significant presence (presence above a
predetermined level) of the third marker (e.g., CK-MB or a
troponin) can indicate with a high degree of diagnostic accuracy
the presence of acute myocardial infarction. In other words, the
test or assay of this invention for detecting a marker of acute
myocardial infarction will distinguish between the following
categories 1 and 2: (1) those who have acute myocardial infarction
and (2) those who do not (i.e., those who have no coronary artery
disease, those who have non-acute coronary artery disease, namely,
stable angina or presumably asymptomatic coronary artery disease,
and those who have unstable angina) but by itself will generally
not be able to distinguish between the non-AMI categories.
[0029] Use of the first and second tests (assays) together on a
patient will allow the patient to be put with a clinically
sufficient degree of diagnostic accuracy into one of three
categories: (1) having no coronary artery disease (first and second
tests negative); (2) having coronary artery disease of the
non-acute type, i.e., either asymptomatic coronary artery disease
or stable angina (first test positive, second test negative); or
(3) having coronary artery disease of the acute type, i.e., either
unstable angina or acute myocardial infarction (both tests
positive). The first and second tests may be used together, for
example, as part of a screening or as part of a routine physical
examination. If the patient is put in the first category, there is
no problem. If the patient is put in the second category, the
physician may take action such as recommending a change in life
style, prescribing appropriate medication, etc. That is
particularly true for asymptomatic CAD patients, who will be placed
in the second category, and who may not have had any previous
indication of coronary artery disease. If the patient is put in the
category of acute coronary disease, the third test of this
invention may be run to determine whether the patient has had or is
having an acute myocardial infarction and, if that is the case, the
physician it may recommend immediate hospitalization and medication
(e.g., tissue plasminogen activator, "TPA").
[0030] Use of the second and third tests (assays) on a patient
without the first test also being run will likely occur less
frequently than use of the first and second tests without the third
test. However, for a patient who has acute symptoms that suggest an
acute myocardial infarction (e.g., chest pain), the physician may
run the third test to determine if the patient is in fact having an
acute myocardial infarction (in which case the third test, e.g.,
for a troponin, would likely be positive) and will likely also want
to run the second test to determine whether the acute myocardial
infarction, if present, is most likely caused by coronary
atherosclerosis (the second test, e.g., for MDA-modified LDL, would
be positive) or if the acute myocardial infarction likely results
from some other cause. For patients presenting classical symptoms
of acute myocardial infarction, use of the second and third tests
together is highly advantageous and the first test might not be
needed in the first instance or at all.
[0031] Thus, in accordance with this invention, if all three tests
are run on a patient, the patient may be placed into one of the
following categories with a clinically sufficient degree of
diagnostic accuracy: (1) having no CAD; (2) having non-acute
(chronic) CAD, namely, either asymptomatic or stable angina; (3)
having the first form of acute CAD, namely, unstable angina; and
(4) having the second form of acute CAD, namely, (a) acute
myocardial infarction ("AMI") that is likely due to atherosclerosis
and (b) AMI that is likely due to a cause other than
atherosclerosis. Categories 2, 3, and 4 may be thought of as being
the stages of coronary artery disease (CAD).
[0032] The clinically significant presence of a first marker in a
sample from a patient (first assay is positive) indicates that the
patient is not in category 1 (no CAD) and is either in category 2
(asymptomatic CAD or stable angina) or 3 (unstable angina) or 4
(AMI). In other words, the clinically significant presence of the
first marker in a sample from the patient indicates that the
patient has coronary artery disease. If the first marker does not
have clinically significant presence in the sample (first assay is
negative), the patient is in category 1, in other words, does not
have CAD. If the assay for the first marker is negative and the
assay for the second marker is positive, it indicates a likely
problem with one or both of the assays because that pairing of test
results is highly unlikely, and one or both tests should be
repeated. Thus, another beneficial feature of the invention is that
by using the first and second assays together, a positive first
assay will confirm a positive second assay, and a negative first
assay will cast significant doubt about a positive second assay and
will thereby indicate a likely problem with one or both assays.
[0033] The clinically significant presence of the first marker in a
sample from a patient coupled with the clinically significant
presence of the second marker in a sample from the patient
indicates that the patient is not in category 1 (no CAD) or 2
(asymptomatic CAD or stable angina) and is instead in category 3
(unstable angina) or category 4 (AMI). If neither the first nor the
second marker has a clinically significant presence, the patient is
in category 1, in other words, does not have CAD.
[0034] The clinically significant presence of the first marker in a
sample from a patient, coupled with the clinically significant
presence of the second marker in a sample from the patient, coupled
with the clinically significant presence of the third marker in a
sample from the patient indicates that the patient is not in
category 1 (no CAD) or 2 (asymptomatic CAD or stable angina) or 3
(unstable angina) and is instead in category 4 (AMI). If the first
and second and third markers do not have clinically significant
presence, the patient is in category 1, in other words, does not
have CAD. If the first and second assays are negative but the third
assay is positive, it indicates AMI caused by something other than
coronary atherosclerotic disease. If the second and third assays
are positive and the first assay is not run (e.g., for a patient
presenting classic AMI symptoms), the patient is in category 4 and
the positive second assay indicates that the heart damage is likely
caused by coronary atherosclerotic disease. If the first test is
negative but the second is positive, the results are equivocal and
it may indicate, e.g., a possible problem with one or more of the
tests. If the first and third tests are positive but the second is
negative, the results are equivocal and it may indicate, e.g., a
possible problem with the tests or a possible non-atherosclerotic
AMI.
[0035] Table I, below, summarizes the possible test outcomes and
resulting categorizations (diagnoses) using the method of this
invention (a plus sign indicates that the test for that marker is
positive; a negative sign indicates that the test for that marker
is negative):
1TABLE I Category First Marker Second Marker Third Marker No CAD -
- - Chronic CAD + - - Unstable Angina + + - AMI (atherosclerotic) +
+ + AMI (non- - - + atherosclerotic) Equivocal - + - Equivocal - +
+ Equivocal + - +
[0036] The "diagnostic accuracy" of a test, assay, or method
concerns the ability of the test, assay, or method to distinguish
between patients having a disease, condition, or syndrome and
patients not having that disease, condition, or syndrome based on
whether the patients have a "clinically significant presence" of an
analyte. By "clinically significant presence" is meant that the
presence of the analyte (e.g., mass, such as milligrams or
nanograms, or mass per volume, such as milligrams per deciliter) in
the patient (typically in a sample from the patient) is higher than
the predetermined cut point (or threshold value) for that analyte
and therefore indicates that the patient has the disease,
condition, or syndrome for which the sufficiently high presence of
that analyte is a marker.
[0037] The terms "high degree of diagnostic accuracy" and "very
high degree of diagnostic accuracy" refer to the test or assay for
that analyte with the predetermined cut point correctly
(accurately) indicating the presence or absence of the disease,
condition, or syndrome. A perfect test would have perfect accuracy.
Thus, for individuals who have the disease, condition, or syndrome,
the test would indicate only positive test results and would not
report any of those individuals as being "negative" (there would be
no "false negatives"). In other words, the "sensitivity" of the
test (the true positive rate) would be 100%. On the other hand, for
individuals who did not have the disease, condition, or syndrome,
the test would indicate only negative test results and would not
report any of those individuals as being "positive" (there would be
no "false positives"). In other words, the "specificity" (the true
negative rate) would be 100%. See, e.g., O'Marcaigh A S, Jacobson R
M, "Estimating The Predictive Value Of A Diagnostic Test, How To
Prevent Misleading Or Confusing Results," Clin. Ped. 1993, 32(8):
485-491, which discusses specificity, sensitivity, and positive and
negative predictive values of a test, e.g., a clinical diagnostic
test.
[0038] Changing the cut point or threshold value of a test (or
assay) usually changes the sensitivity and specificity but in a
qualitatively inverse relationship. For example, if the cut point
is lowered, more individuals in the population tested will
typically have test results over the cut point or threshold value.
Because individuals who have test results above the cut point are
reported as having the disease, condition, or syndrome for which
the test is being run, lowering the cut point will cause more
individuals to be reported as having positive results (i.e., that
they have the disease, condition, or syndrome). Thus, a higher
proportion of those who have the disease, condition, or syndrome
will be indicated by the test to have it. Accordingly, the
sensitivity (true positive rate) of the test will be increased.
However, at the same time, there will be more false positives
because more people who do not have the disease, condition, or
syndrome (i.e., people who are truly "negative") will be indicated
by the test to have analyte values above the cut point and
therefore to be reported as positive (i.e., to have the disease,
condition, or syndrome) rather than being correctly indicated by
the test to be negative. Accordingly, the specificity (true
negative rate) of the test will be decreased. Similarly, raising
the cut point will tend to decrease the sensitivity and increase
the specificity. Therefore, in assessing the accuracy and
usefulness of a proposed medical test, assay, or method for
assessing a patient's condition, one should always take both
sensitivity and specificity into account and be mindful of what the
cut point is at which the sensitivity and specificity are being
reported because sensitivity and specificity may vary significantly
over the range of cut points.
[0039] There is, however, an indicator that allows representation
of the sensitivity and specificity of a test, assay, or method over
the entire range of cut points with just a single value. That
indicator is derived from a Receiver Operating Characteristics
("ROC") curve for the test, assay, or method in question. See,
e.g., Shultz, "Clinical Interpretation Of Laboratory Procedures,"
chapter 14 in Teitz, Fundamentals of Clinical Chemistry, Burtis and
Ashwood (eds.), 4th edition 1996, W.B.Saunders Company, pages
192-199; and Zweig et al., "ROC Curve Analysis: An Example Showing
The Relationships Among Serum Lipid And Apolipoprotein
Concentrations In Identifying Patients With Coronory Artery
Disease," Clin. Chem., 1992, 38(8): 1425-1428.
[0040] An ROC curve is an x-y plot of sensitivity on the y-axis, on
a scale of zero to one (i.e., 100%), against a value equal to one
minus specificity on the x-axis, on a scale of zero to one (i.e.,
100%). In other words, it is a plot of the true positive rate
against the false positive rate for that test, assay, or method. To
construct the ROC curve for the test, assay, or method in question,
patients are assessed using a perfectly accurate or "gold standard"
method that is independent of the test, assay, or method in
question to determine whether the patients are truly positive or
negative for the disease, condition, or syndrome (for example,
coronary angiography is a gold standard test for the presence of
coronary atherosclerosis). The patients are also tested using the
test, assay, or method in question, and for varying cut points, the
patients are reported as being positive or negative according to
the test, assay, or method. The sensitivity (true positive rate)
and the value equal to one minus the specificity (which value
equals the false positive rate) are determined for each cut point,
and each pair of x-y values is plotted as a single point on the x-y
diagram. The "curve" connecting those points is the ROC curve.
[0041] The area under the curve ("AUC") is the indicator that
allows representation of the sensitivity and specificity of a test,
assay, or method over the entire range of cut points with just a
single value. The maximum AUC is one (a perfect test) and the
minimum area is one half. The closer the AUC is to one, the better
is the accuracy of the test.
[0042] By a "high degree of diagnostic accuracy" is meant a test or
assay (such as the test of the invention for determining the
clinically significant presence of the third analyte, which thereby
indicates the presence of an acute myocardial infarction) in which
the AUC (area under the ROC curve for the test or assay) is at
least 0.70, desirably at least 0.75, more desirably at least 0.80,
preferably at least 0.85, more preferably at least 0.90, and most
preferably at least 0.95.
[0043] By a "very high degree of diagnostic accuracy" is meant a
test or assay (such as the test of the invention for determining
the clinically significant presence of the first analyte, which
thereby indicates the presence of coronary artery disease, or the
test for determining the clinically significant presence of the
second analyte, which thereby indicates the presence of an acute
stage of coronary artery disease) in which the AUC (area under the
ROC curve for the test or assay) is at least 0.875, desirably at
least 0.90, more desirably at least 0.925, preferably at least
0.95, more preferably at least 0.975, and most preferably at least
0.98.
[0044] By a "clinically sufficient degree of diagnostic accuracy"
is meant a method (such as the method of the invention) that (1) in
a first test can assay for a first marker whose presence above a
predetermined level can indicate with a very high degree of
diagnostic accuracy the presence of coronary artery disease, (2) in
a second test can assay for a second marker whose presence above a
predetermined level can indicate with a very high degree of
diagnostic accuracy the presence of an acute stage of coronary
artery disease, and (3) in a third test can assay for a third
marker whose presence above a predetermined level can indicate with
a high degree of diagnostic accuracy the presence of acute
myocardial infarction; wherein at least the second test is run and
either or both of the first and third tests are run.
[0045] The method of the present invention provides a degree of
clinical diagnostic accuracy for detecting the presence of and for
distinguishing between or among the non-acute (chronic) and the
acute stages of coronary artery disease for a human patient from
the general population that is that is significantly higher than
any other previously known method. However, the advantages of this
invention include not just the high overall accuracy made possible
by its tests but that the use of the tests together rapidly
provides all the information needed by the clinician about the
patient to permit possible life-saving treatment. Thus, the
physician will know by using the method of this invention for a
specific patient that the patient does not have coronary artery
disease; or, if the presence of the disease is already known, that
the measures being taken to deal with it are either adequate or
inadequate; or that patient has the disease but did not know it and
that a change in diet and/or exercise habits and/or medication
and/or other treatment are needed, but not on an emergency basis;
or that the patient has a life-threatening coronary problem and
must be dealt with on an emergency basis. Another advantage is that
acute myocardial infarctions due to coronary atherosclerosis can be
distinguished from those due to other causes, which knowledge will
significantly affect treatment. Yet another advantage is that in
some cases the tests will act to confirm the validity of each other
and thereby give the physician more confidence in diagnosis and
treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Lipoproteins are multicomponent complexes of protein and
lipids. Each type of lipoprotein has a characteristic molecular
weight, size, chemical composition, density, and physical role. The
protein and lipid are held together by noncovalent forces.
[0047] Lipoproteins can be classified on the basis of their density
as determined by ultracentrifugation. Thus, four classes of
lipoproteins can be distinguished: High Density Lipoproteins
("HDL"), Intermediate Density Lipoproteins ("IDL"), Low Density
Lipoproteins ("LDL"), and Very Low Density Lipoproteins
("VLDL").
[0048] The purified protein components of a lipoprotein particle
are called apolipoproteins (apo). Each type of lipoprotein has a
characteristic apolipoprotein composition. In LDL the prominent
apolipoprotein protein is apo B-100, which is one of the longest
single chain polypeptides known and consists of 4536 amino acids.
Of these amino acids the lysine residues or moieties (there are 356
such lysine residues or moieties) can be substituted or modified by
aldehydes (e.g., malondialdehyde).
[0049] Oxidation of the lipids in LDL (whether in vitro, e.g., by
copper-induced oxidation, or whether in vivo) results in the
generation of reactive aldehydes, which can then interact with the
lysine residues or moieties of apo B-100. The outcome of this
lysine substitution or modification is that the resulting oxidized
low density lipoprotein ("OxLDL"), which is also
malondialdehyde-modified low density lipoprotein ("MDA-modified
LDL"), is no longer recognized by the LDL receptor at the surface
of fibroblasts but by scavenger receptors at the surface of
macrophages. At least 60 out of the 356 lysines (or lysine residues
or moieties) of apo B-100 have to be substituted in order to be
recognized by the scavenger receptors. The uptake of such OxLDL by
macrophages results in foam cell generation, which is considered to
be an initial step in atherosclerosis.
[0050] Endothelial cells under oxidative stress (e.g., in acute
myocardial infarction patients) and activated blood platelets also
produce aldehydes, which interact with the lysine moieties in apo
B-100, resulting in the generation of aldehyde-modified LDL that is
also recognized by the scavenger receptors. However, the lipids in
this aldehyde-modified LDL are not oxidized. Enzymatic activity in
macrophages (e.g., myeloperoxidase) results in the oxidation of
both the lipid and the protein moieties of LDL. All these pathways
result in aldehyde-type modification of the protein moiety of
LDL.
[0051] The first marker can be any marker whose clinically
significant presence indicates with a very high degree of
diagnostic accuracy the presence of coronary artery disease.
Desirably, the first marker is an atherogenic protein. Preferably,
the atherogenic protein comprises OxLDL containing at least 60,
desirably up to about 90, more desirably up to about 120,
preferably up to about 180, more preferably up to about 210, and
most preferably possibly up to about 240 substituted lysine
residues per apo B-100 moiety.
[0052] The second marker can be any marker whose clinically
significant presence indicates with a very high degree of
diagnostic accuracy the presence of an acute stage of coronary
artery disease. Desirably, the second marker is an atherogenic
protein. Preferably, the atherogenic protein comprises MDA-modified
LDL containing at least 60, desirably up to about 90, more
desirably up to about 120, preferably up to about 180, more
preferably up to about 210, and most preferably possibly up to
about 240 substituted lysine residues per apo B-100 moiety.
[0053] The third marker can be any marker whose clinically
significant presence indicates with a high degree of diagnostic
accuracy the presence of acute myocardial infarction. The gold
standard chemical marker for acute myocardial infarction has been
CK-MB but may be shifting to the troponins (e.g., troponin I,
troponin T). See, e.g., Adams et al., "Cardiac Troponin I, A Marker
With High Specificity For Cardiac Injury," Circulation 1993, 88(1):
101-106; Antman et al., "Cardiac-Specific Troponin I Levels To
Predict The Risk Of Mortality In Patients With Acute Coronary
Syndromes," N. Eng. J. Med. 1996, 335(18): 1342-1349; Hamm et al.,
"Emergency Room Triage Of Patients With Acute Chest Pain By Means
Of Rapid Testing For Cardiac Troponin T Or Troponin I," N. Eng. J.
Med. 1997, 337(23): 1648-1653; Ohman et al., "Cardiac Troponin T
Levels For Risk Stratification In Acute Myocardial Ischemia," N.
Eng. J. Med. 1996, 335(18): 1333-1341; and Van de Werf, "Cardiac
Troponins In Acute Coronary Syndromes," N. Eng. J. Med. 1996,
335(18): 1388-1389. Another substance that may possibly be used as
the third marker is a marker for active or incipient coronary
thrombosis. Thus, a "marker whose presence above a predetermined
level can indicate with a high degree of diagnostic accuracy the
presence of acute myocardial infarction" should be understood to
include markers of active and incipient coronary thrombosis even
before substances indicative of cardiac tissue damage or death have
been formed and/or released. Generally speaking, the third marker
will typically be a "heart protein," which as used herein means a
protein (e.g., an enzyme) that is produced as a result of or is
otherwise associated with ischemic damage to the heart or that is a
precursor or derivative of that protein.
[0054] Testing for the clinically significant presence of the
markers may use any assays, methodology, and equipment provided the
benefits of this invention can be achieved, e.g., chemical assays
and immunological assays, such as competitive and sandwich assays,
may be used. "Competitive assays" are well-known and any
competitive assay may be used in this invention provided the
benefits of the invention can be achieved. "Sandwich assays" are
well-known and any sandwich assay may be used in this invention
provided the benefits of the invention can be achieved.
[0055] In an immunological assay, any antibodies that have suitably
high affinity for the target species may be used, and preferably
the antibodies are monoclonal antibodies. As used herein, "high
affinity" means an affinity constant (association constant) of at
least about 5.times.10.sup.8 M.sup.-1 (where "M" indicate molarity
or moles per liter, and "M.sup.-1" indicates reciprocal molarity or
liters per mole), desirably of at least about 1.times.10.sup.9
M.sup.-1, preferably of at least about 1.times.10.sup.10 M.sup.-1,
and most preferably of at least about 1.times.10.sup.11 M.sup.-1.
As used herein, "low affinity" (in contradistinction to high
affinity) means an affinity constant (association constant) of less
than about 1.times.10.sup.5 M.sup.-1, desirably less than about
1.times.10.sup.6 M.sup.-1, and preferably less than about
1.times.10.sup.5 M.sup.-1. Affinity constants are determined in
accordance with the appropriate method described in Holvoet,
Collen, et al., J. Clin. Invest. 1994, 93: 89-98.
[0056] The preferred antibodies used in this first and second
assays of this invention will bind with OxLDL and/or MDA-modified
LDL whose apo B-100 moieties contain at least 60, 20 desirably up
to about 90, more desirably up to about 120, preferably up to about
180, more preferably up to about 210, and most preferably possibly
up to about 240 substituted lysine residues per apo B-100 moiety.
The range of lysine substitution will generally be from 60 up to
about 240 substituted lysine moieties per apo B-100 moiety and
sometimes from 60 up to about 180 substituted lysine moieties per
apo B-100 moiety.
[0057] Each monoclonal antibody used in the first and second assays
is desirably highly specific for a conformational epitope that is
present when at least about 60, preferably up to about 120 lysine
residues, are substituted and by virtue thereof can distinguish the
first and second markers of the first and second assays. Antibodies
recognizing epitopes present when less than about 60 lysines per
apo B-100 moiety are substituted or modified are less specific but
are still useful (e.g., they may be used as the secondary antibody
in a sandwich ELISA).
[0058] The preferred antibodies used herein are monoclonal
antibodies mAb-4E6, mAb-1h11, and mAb-8A2. Their affinity constants
for native LDL, MDA-modified LDL, and OxLDL are as follows (the
units are liters per mole, which equals the reciprocal of molarity
or M.sup.-):
2TABLE II Antibody Native LDL MDA-modified LDL O .times. LDL
mAb-4E6 less than 1 .times. 10.sup.6 3 .times. 10.sup.10 2 .times.
10.sup.10 mAb-1H11 less than 1 .times. 10.sup.6 3 .times. 10.sup.10
less than 1 .times. 10.sup.6 mAb-8A2 5 .times. 10.sup.9 1 .times.
10.sup.10 1 .times. 10.sup.10
[0059] Monoclonal antibody mAb-4E6 is produced by hybridoma Hyb4E6
deposited at the BCCM under deposit accession number LMBP 1660 CB
on or about Apr. 24, 1997. Monoclonal antibody mAb-1H11 is produced
by hybridoma Hyb1H11 deposited at the BCCM under deposit accession
number LMBP 1659 CB on or about Apr. 24, 1997. Monoclonal antibody
mAb-8A2 is produced by hybridoma Hyb8A2 deposited at the BCCM under
deposit accession number LMBP 1661 CB on or about April 24,
1997.
[0060] The BCCM is the Belgian Coordinated Collections of
Microorganisms authorized by the Budapest Treaty Of Apr. 28, 1977
On The International Recognition Of The Deposit Of Microorganisms
For The Purpose Of Patent Procedure ("Budapest Treaty"). Its
address is c/o The University of Gent, K. L. Ledeganckstraat 35,
B-9000 Gent, Belgium.
[0061] The three deposits were made at the BCCM under conditions
prescribed by the Budapest Treaty. In accordance with The United
States Code Of Federal Regulations (see 37 CFR .sctn. 1.808) and
The United States Patent And Trademark Office's Manual Of Patent
Examination ("MPEP") (see .sctn. 2410.01), all restrictions imposed
by the depositor on the availability to the public of the deposited
material (except as permitted by the MPEP) will be irrevocably
removed upon the granting of any patent issuing from this
application or from any continuing application based thereon.
[0062] As described elsewhere, those three monoclonal antibodies
were obtained in the following way (see PCT Applications
PCT/EP97/03287, filed Jun. 20, 1997, and PCT/EP97/03493, filed Jul.
1, 1997 [both designating the United States and other countries,
and naming Paul Holvoet and Dsir Collen as inventors, and, as noted
above, both of which are incorporated herein in there entireties
for all purposes]).
[0063] Balb/c mice were immunized by intravenous and
intraperitoneal injection of either OxLDL or MDA-modified LDL.
OxLDL was obtained by in vitro incubation of LDL (final apo B-100
concentration 700 .mu.g/mL) with copper chloride (final
concentration 640 .mu.M) for 16 hours at 37.degree. C. MDA-modified
LDL was prepared by incubation of LDL (final apo B-100
concentration 700 .mu.g/mL) with a 0.25 M MDA-solution for 3 hours
at 37.degree. C. The numbers of substituted lysines, measured in
the TBARS assay, was typically 210 per apo B-100 molecule for OxLDL
and 240 for MDA-modified LDL. Hybridomas were obtained by
PEG-induced fusion of spleen lymphocytes derived from immunized
mice with P3-X63/Ag-6.5.3 myeloma cells according to standard
techniques (Holvoet, Collen, et al., J. Clin. Invest. 1994; 93:
89-98). The screening for hybridomas producing specific antibodies
was performed with ELISA using microtiter plates coated with
MDA-modified LDL or copper-oxidized LDL. Three hundred eight
hybridomas were obtained after immunization of mice with either
OxLDL (211) or MDA-modified LDL (97). Hyb4E6 produced antibodies
specific for both MDA-modified and copper-oxidized LDL (mAb-4E6),
and Hyb1H11 produced antibodies specific for MDA-modified LDL
(mAb-1H11) alone. Mice immunized with LDL in a similar method
yielded hybridoma Hyb8A2, which produced antibody mAb-8A2.
[0064] The preferred assay is an Enzyme-Linked Immunosorbent Assay
("ELISA"). For example, in the case of a competitive ELISA, a solid
substrate coated with OxLDL or MDA-modified LDL may be contacted
for a predetermined period of time with the monoclonal antibody
mAb-4E6 and a sample thought or known to contain OxLDL and/or
MDA-modified LDL, after which period of time unbound antibody and
sample are removed and a binding reaction between antibody and
OxLDL and/or MDA-modified LDL bound to the substrate is visualized
and/or quantified. Quantification in a competitive ELISA is
indirect because the binding between the antibody and the analyte
in the sample is not measured but instead the amount of antibody
that binds to the known amount of OxLDL or MDA-modified LDL that is
coated on (bound to) the substrate is measured. The more antibody
bound to the known amount of OxLDL or MDA-modified LDL coated on
the substrate, the less analyte there was in the sample.
[0065] A typical competitive assay using monoclonal antibody
mAb-4E6 is as follows.
[0066] It is based on the inhibition by copper-oxidized LDL of the
binding of mAb-4E6 to the coated wells of microtiter plates. Thus,
standard OxLDL (or MDA-modified LDL) and plasma samples are diluted
in PBS (phosphate buffered saline) containing 1 mM EDTA, 20 pM
Vitamin E, 10 .mu.M butylated hydroxytoluene, 20 PM dipyridamole,
and 15 mM theophylline to prevent in vitro LDL oxidation and
platelet activation. Equal volumes of diluted purified mAb-4E6
solution (final concentration 7.5 ng/mL) and of either diluted
standard solution or diluted plasma samples (copper-oxidized LDL
added as competing ligand at a final concentration ranging from 50
to 500 ng/mL) are mixed and incubated for 30 min at room
temperature. Then 200 pL aliquots of the mixtures are added to
wells coated with MDA-modified LDL or OxLDL. The aliquots are
incubated for 2 hours at room temperature. After washing, the wells
are incubated for 1 h with horse-radish peroxidase conjugated
rabbit IgG raised against mouse immunoglobulins and washed again.
The peroxidase reaction is performed (see Holvoet, Collen, et al.,
J. Clin. Invest. 1995, 95: 2611-2619) and the absorbance (A) is
read at 492 nm. Controls without competing ligand and blanks
without antibody may be routinely included. The percent inhibition
of binding of mAb-4E6 to the immobilized ligand may be calculated
as: 1 A 492 nm control - A 492 nm sample A 492 nm control - A 492
nm blank
[0067] and standard curves may be obtained by plotting the
percentage of inhibition against the concentration of competing
ligand. The lower limit of detection is 0.020 mg/dL in undiluted
human plasma.
[0068] In the case of a sandwich ELISA, mAb-4E6 (for MDA-modified
LDL and OxLDL) or mAb-1H11 (for MDA-modified LDL) may be bound to a
solid substrate and subsequently contacted with a sample to be
assayed. After removal of the sample, binding between the specific
antibody and OxLDL and/or MDA-modified LDL captured out of the
sample can be visualized and/or quantified by detection means.
Detection means may be a labeled, less specific secondary antibody
that recognizes a different part of the apo B-100 moiety of the
captured analyte (e.g., mAb-8A2).
[0069] A typical sandwich assay using monoclonal antibodies mAb-4E6
and mAb-8A2 is as follows. It is based on the binding of
immunoreactive material to the wells of microtiter plates coated
with the monoclonal antibody mAb-4E6 and the detection of bound
immunoreactive material with the use of the monoclonal antibody
mAb-8A2 labeled with peroxidase. This version of the ELISA is more
suited for use in the clinical laboratory because it overcomes the
need to prepare standard solutions of in vitro oxidized and/or
aldehyde-modified LDL.
[0070] Standard preparations and plasma samples are diluted in PBS
containing antioxidants and antiplatelet agents as described above
in connection with the competitive ELISA, 180 .mu.L aliquots of
80-fold diluted plasma and of standard solutions containing between
10 and 0.01 nM of MDA-modified LDL are applied to the wells of
microtiter plates coated with mAb-4E6 (200 .mu.L of a 4 .mu.g/mL
IgG solution), and incubated for 2 hours at room temperature. After
washing, the wells are incubated for 1 hour with horseradish
peroxidase conjugated mAb-8A2, IgG (final IgG concentration 65
ng/mL), and washed again. The peroxidase reaction is performed as
described above in connection with the competitive ELISA. The
absorbance measured at 492 nm will correlate with the log-value of
the MDA-modified LDL concentration in the range between 1.5 nM and
0.3 nM.
[0071] Tests for the third marker (e.g., CK-MB, troponin I) are
known. See, e.g., Adams et al., Circulation 1993, 88(1): 101-106;
Antman et al., N. Eng. J. Med. 1996, 335(18): 5 1342-1349; Hamm et
al., N. Eng. J. Med. 1997, 337(23): 1648-1653; Ohman et al., N.
Eng. J. Med. 1996, 335(18): 1333-1341; and Van de Werf, N. Eng. J.
Med. 1996, 335(18): 1388-1389.
[0072] As used herein, "a human patient from the general
population" should be understood broadly to be any human being and
is not limited to human beings who have been formally admitted to
hospitals or who do or do not have specific diseases, conditions,
or syndromes. There may possibly be one or more subgroups of the
general population for which the method of this invention is not as
desirable; however, what those one or more subgroups are (if they
exist at all) is not known at present.
[0073] The "sample from the patient" used herein may be any sample
that allows the benefits of this invention to be achieved.
Typically, the "sample from the patient" will be a fluid sample,
typically whole blood or a fluid derived from whole blood (such as
plasma or serum). Fluid samples (particularly whole blood, plasma,
or serum), as opposed to tissue samples, have the advantage of
being easily and quickly obtained and tested, which is particularly
important in a clinical setting where time may be of the essence.
Also, clinicians are accustomed to withdrawing fluid samples
(particularly blood) from patients, and some of the markers may not
be present or may not be present in sufficient quantities in tissue
samples.
[0074] Whole blood may contain substances, e.g., cells, that
interfere with the tests used in the method of the invention and,
therefore, whole blood is a less preferred sample. The preferred
sample is plasma, which is whole blood from which the cells (red
blood cells, white blood cells, and platelets) have been removed,
e.g., by centrifugation. Serum is plasma from which the fibrinogen
has been removed (e.g., by causing clotting and then removing the
clotted material) and is also less preferred than plasma.
[0075] As indicated above, any assays, methodology, and equipment
may be used provided the benefits of this invention can be
achieved. Thus, for example, the invention is not limited to the
use of microwell plate technology. If, for example, the tests of
the method of this invention involve using antibodies, those
antibodies may be used in a wide variety of automated immunologic
assay systems, which include chemiluminescent immunoassay systems,
microparticle enzyme immunoassay systems, fluorescence polarization
immunoassay systems, and radioimmunoassay systems.
[0076] The method of this invention was used in connection with
almost three hundred patients from the general population (who in
this case did not include heart transplant individuals), as
described below. Broadly speaking, statistical analyses of the
results indicated that of the possible markers tested, the best
marker for the first test was OxLDL, that the best marker for the
second test was MDA-modified LDL, and that the best marker for the
third test was troponin I.
[0077] A total of 286 individuals associated with the University
Hospital Of Leuven either as employees or as individuals who were
brought to the emergency department and/or admitted to the Hospital
were studied: 105 patients with acute coronary syndromes, 64
patients with stable CAD, and 117 controls.
[0078] Individuals were classified as having an acute coronary
syndrome (i.e., having an acute stage of coronary artery disease)
if they had ischemic chest discomfort with ST-segment elevation or
depression of more than 0.5 mm or T wave inversion of more than 1
mm. Of the individuals having an acute stage of coronary artery
disease, those whose elevated creatine kinase (CK)-MB levels (and
at least 3% of total CK) were present at the time of admission or
in samples taken at 6 to 8 hours after admission were classified as
having AMI. Alternatively, those acute-stage individuals who had no
such CK-MB elevations were classified as having unstable
angina.
[0079] Individuals with angiographically documented CAD and no
clinical signs of ischemia within the previous month were
considered to have stable CAD (i.e., in this case, stable
angina).
[0080] One hundred seventeen individuals (72 males/45 females; mean
age=55 years) without a history of atherosclerotic cardiovascular
disease were used as controls. They were selected from laboratory
and clinical staff of the Hospital and from a population of
individuals admitted to the Hospital who did not have a history of
atherosclerotic cardiovascular disease.
[0081] Venous blood samples were taken in the fasting state in
controls and in individuals with stable angina. In individuals with
acute coronary syndromes, blood samples were taken on admission
before the start of treatment. Blood samples were collected on 0.01
M citrate, containing 1 mM EDTA, 20 .mu.M vitamin E, 10 .mu.M
butylated hydroxytoluene, 20 .mu.M dipyridamole, and 15 mM
theophylline to prevent in vitro LDL oxidation and platelet
activation. Blood samples were centrifuged at 3,000 g for 15
minutes at room temperature within 1 hour of collection and the
resulting plasma was stored at -20.degree. C. until the assays were
performed.
[0082] LDL were isolated from pooled plasma of fasting
normolipidemic donors by density gradient ultracentrifugation
(Havel et al., J. Clin. Invest. 1955, 34: 1345-1353). MDA-modified
LDL and copper-oxidized LDL were prepared as described in Haberland
et al., Proc. Natl. Acad. Sci USA. 1982, 79: 1712-1716, and
Steinbrecher, J. Biol. Chem. 1987, 262(8): 3603-3608, and were used
as standards. Characterization of modified LDL involved measurement
of thiobarbituric acid reactive substances ("TBARS"), determination
of electrophoretic mobility on 1% agarose gels, quantitation of
cholesterol and fatty acids by HPLC on a Nova-Pak C-1 8
reversed-phase column (Waters Associates, Milford, Mass.),
quantitation of proteins by Lowry assay, and of phospholipids by
enzymatic assay (Biomerieux, Marcy, France). See Holvoet, Collen,
et al., Arterioscler. Thromb. Vasc. Biol. 1998, 18(1): 100-107, and
Holvoet, Collen, et al. J. Clin. Invest. 1995, 95: 2611-2619. Apo
B1-100 molecules of in vitro MDA-modified LDL and of
copper-oxidized LDL contained on average 244 and 210 substituted
lysines, respectively. As noted above, although the extent of
lysine substitution of in vitro MDA-modified LDL and
copper-oxidized LDL is very similar, the lipid moiety in
MDA-modified LDL is not oxidized.
[0083] A mAb-4E6 based ELISA was used for the quantitation of OxLDL
in plasma (see Holvoet, Collen, et al., Arterioscler. Thromb. Vasc.
Biol. 1998, 18(1): 100-107; Holvoet, Collen, et al., Thromb.
Haemost. 1996, 76(5): 663-669; Holvoet and Collen, Arterioscler.
Thromb. Vasc. Biol. 1997, 17(11): 2376-2382; and Holvoet, Collen,
et al., Arterioscler. Thromb. Vasc. Biol. 1998, 18: 415-422). This
monoclonal antibody allows the detection of 0.025 mg/dL
MDA-modified LDL or copper-oxidized LDL in the presence of 500
mg/dL native LDL. Plasma levels of MDA-modified LDL were measured
in a mAb-1H11 based ELISA (see Holvoet, Collen, et al., J. Clin.
Invest. 1995, 95: 2611-2619). This monoclonal antibody allows the
detection of 0.025 mg/dL MDA-modified LDL, but not of
copper-oxidized LDL, in the presence of 500 mg/dL native LDL.
Because the specificities of the two antibodies depend on the
extent of protein modification, all lipoprotein concentrations are
expressed in terms of protein.
[0084] Total cholesterol, HDL cholesterol, and triglycerides were
measured by enzymatic methods (Boehringer Mannheim, Meylon,
France). LDL cholesterol values were calculated with the Friedewald
formula. Troponin I levels were measured on a Beckman ACCESS
immunoanalyzer using commercially available monoclonal antibodies
(Sanofi, Toulouse, France). C-reactive protein levels were measured
in a commercial immunoassay (Boehringer, Brussels, Belgium), and
plasma levels of D-dimer were measured in an ELISA as described
previously (see Declerck, Holvoet, Collen, et al., Thromb. Haemost.
1987, 58(4): 1024-1029). C-reactive protein is a marker of
inflammation. D-dimer is a marker for thrombotic syndromes.
[0085] The values obtained are shown in Table III, below ("n"
indicates the number of individuals independently known to be in
each category).
3 TABLE III Controls Stable angina Unstable angina AMI (n = 117) (n
= 64) (n = 42) (n = 63) Age 55 .+-. 11 65 .+-. 10 72 .+-. 12 63
.+-. 11 Male/female ratio 72/45 53/11 28/14 42/21 Total cholesterol
(mg/dL) 180 .+-. 31 180 .+-. 35.3 175 .+-. 36.9 175 .+-. 37.2 LDL
cholesterol (mg/dL) 110 .+-. 26 115 .+-. 30 109 .+-. 33.4 111 .+-.
32.4 HDL cholesterol (mg/dL) 49 .+-. 18 37.6 .+-. 13.2 45.2 .+-.
15.6 37.5 .+-. 9.7 Triglycerides (mg/dL) 137 .+-. 66 123 .+-. 46.2
103 .+-. 55.4 125 .+-. 56.7 Oxidized LDL (mg/dL) 0.85 .+-. 0.54
2.65 .+-. 0.97 3.22 .+-. 0.85 2.97 .+-. 1.02 MDA-modified LDL
(mg/dL) 0.39 .+-. 0.15 0.46 .+-. 0.20 1.07 .+-. 0.28 1.19 .+-. 0.43
Troponin I (ng/mL) 0.0092 .+-. 0.011 0.035 .+-. 0.12 0.37 .+-. 0.66
1.30 .+-. 1.08 C-reactive protein (mg/dL) 3.38 .+-. 1.79 6.28 .+-.
9.0 17.4 .+-. 29.8 18.2 .+-. 35.5 D-dimer (.mu.g/dL) 166 .+-. 162
299 .+-. 208 367 .+-. 340 602 .+-. 632 Quantitative data represent
means .+-. standard deviations. "AMI" is acute myocardial
infarction.
[0086] Plasma levels of OxLDL were 0.85.+-.0.54 mg/dL
(mean.+-.standard deviation) in the 117 controls, and were 3.1-fold
higher (p<0.001) in the 64 patients with stable angina pectoris,
3.8-fold higher (p<0.001) in the 42 patients with unstable
angina pectoris, and 3.5-fold higher (p<0.001) in the 63
patients with AMI. (For comparison, a group of 79 heart transplant
patients 5 without CAD had OxLDL of 1.27.+-.0.061 mg/dL or 1.5-fold
higher than the 117 controls and a group of 28 heart transplant
patients with stable CAD had OxLDL of 2.49.+-.0.18 mg/dL or
2.9-fold higher than the controls. The reason for the apparent
difference between the values for the non-CAD individuals who have
had or have not had heart transplants is not known with certainty.)
These results show that the test or assay of this invention used
for detecting a marker of coronary artery disease in a patient in
the general population will distinguish with a very high degree of
diagnostic accuracy between the following categories 1 and 2: (1)
those who do not have coronary artery disease and (2) those who do
have one of the categories or stages of coronary artery disease,
but by itself is not able to distinguish with a sufficient degree
of accuracy between the categories (or stages) of coronary artery
disease.
[0087] Plasma levels of MDA-modified LDL were 0.39.+-.0.15 mg/dL in
the 117 controls, were only 1.2-fold higher in the 64 patients with
stable angina pectoris, but were 2.7-fold higher (p<0.001) in
the 42 patients with unstable angina pectoris and 3.1-fold higher
(p<0.001) in the 63 AMI patients. (For comparison, a group of 79
heart transplant patients without CAD had MDA-modified LDL of
0.38.+-.0.016 mg/dL or essentially the same as the 117 controls and
a group of 28 heart transplant patients with stable CAD had
MDA-modified LDL of 0.39.+-.0.038 mg/dL or also essentially the
same as the controls.) These results show that the test or assay of
this invention for detecting a marker of an acute stage of coronary
artery disease will distinguish between the following categories 1
and 2: (1) those who do not have an acute stage of coronary artery
disease (i.e., those who have either (a) no coronary artery disease
or have non-acute coronary artery disease, namely, (b) asymptomatic
coronary artery disease or (c) stable angina) but by itself will
generally not be able to distinguish with a sufficient degree of
accuracy between those three categories a, b, and c, and (2) those
who do have one of the two categories or stages of acute coronary
artery disease (i.e., those who have either (a) unstable angina or
(b) acute myocardial infarction) but by itself will generally not
be able to distinguish with a sufficient degree of accuracy between
the two acute categories.
[0088] Plasma levels of troponin I were 0.0092.+-.0.011 ng/mL in
the 117 controls, were only 3.8-fold higher in the 64 patients with
stable angina, but were 40-fold higher (p<0.001) in the 42
patients with unstable angina and 141-fold higher (p<0.001) in
the 63 AMI patients. In agreement with previously published data,
troponin I was found to be a marker of acute myocardial infarction
(see Adams et al., Circulation, 1993, 88(1): 101-106; and Antman et
al., N. Eng. J. Med. 1996, 335(18): 1342-1349).
[0089] Plasma levels of C-reactive protein were 3.38.+-.1.79 mg/dL
in the 117 controls, were only 1.9-fold higher in the 64 patients
with stable angina, but were 5.1-fold higher (p<0.001) in the 42
patients with unstable angina and 5.4-fold higher in the 63 AMI
patients (p<0.001). In agreement with previously published data,
C-reactive protein was found to be a marker of acute coronary
syndromes (see Muldoon et al., Ryan et al., Oltrona et al., and
Liuzzo et al., letters and reply by authors, N. Engl. J. Med. 1995,
332(6): 398-400).
[0090] Plasma levels of D-dimer were 166.+-.162 .mu.g/dL in the 117
controls, were only 1.8-fold higher in the 64 patients with stable
angina, but were 2.2-fold higher (p<0.001) in the 42 patients
with unstable angina and 3.6-fold higher in the 63 AMI patients
(p<0.001). In agreement with earlier published data, D-dimer was
found to be a marker of acute coronary syndromes (Hoffineister,
Circulation 1995, 91(10): 2520-2527).
[0091] The data were also analyzed to determine the sensitivity and
specificity of OxLDL (cut-point of 1.4 mg/dL), MDA-modified LDL
(cut-point of 0.7 mg/dL), and troponin I (cut-point of 0.07 ng/mL)
for the individual stages of coronary artery disease. In other
words, below the cut-point, the individual is classified as not
having the stage of CAD in question, and at or above the cut-point,
the individual is classified as having that stage of CAD. The
sensitivities and specificities are shown in Table IV as
follows.
4TABLE IV For Acute Myocardial Infarction: O .times. LDL
Sensitivity = 97% Specificity = 100% MDA-modified LDL Sensitivity =
94% Specificity = 94% Troponin I Sensitivity = 90% Specificity =
94% For Unstable Angina: O .times. LDL Sensitivity = 100%
Specificity = 100% MDA-modified LDL Sensitivity = 95% Specificity =
94% Troponin I Sensitivity = 33% Specificity = 94% For Stable
Angina: O .times. LDL Sensitivity = 94% Specificity = 100%
MDA-modified LDL Sensitivity = 7.8% Specificity = 97% Troponin I
Sensitivity = 6.3% Specificity = 100%
[0092] The data were compared using nonparametric Kruskal-Wallis
ANOVA followed by Dunnet's multiple comparison test using the Prism
statistical program (Graph Pad Software, San Diego, Calif.). Plasma
levels of OxLDL and of MDA-modified LDL in patients with normal or
elevated levels of troponin I, C-reactive protein, or D-dimer, and
in patients with and without peripheral vascular disease were
compared by Mann-Whitney test. Discontinuous parameters were
compared by Chi-square analysis.
[0093] A logistic regression model was used to describe
univariately the relation between CAD (yes or no, i.e., does the
individual have CAD or not) and several covariates. For the
individuals who had CAD, the relation between the stability of the
CAD (stable or unstable) and the covariates was checked by logistic
regression models. For the individuals who had an unstable CAD, the
relation between unstable angina or AMI and the covariates was
checked by logistic regression models. The relations between (1)
stable or unstable angina, (2) stable angina or AMI, and (3) stable
angina, unstable angina, or AMI and the covariates were examined by
(multigroup for the latter) logistic regression models. For
continuous variables, cubic spline functions were used to model the
relationship between the covariates and the response. This allowed
specifying non-linear functions of the predictors in the model. A
multiple logistic regression model was fitted, including all
univariately significant variables and their confounding factors.
The confounding factors were checked by means of a Spearman
correlation coefficient. The measure of predictive discrimination
used to characterize the model performance was the area under the
Receiver Operating Characteristic (ROC) curve. The software used
was FE Harell Jr., "Design, S Functions For
Biostatistical/Epidemiologic Modeling, Testing, Estimation,
Validation, Graphics, And Prediction" (available from
statlib.cmu.edu; request "send design from S," 1994); S-plus.RTM.
4.0 Release 3 for Windows (Mathsoft Inc., Cambridge, Mass., USA);
and SAS/STAT software version 6.12: SAS Institute Inc. (Cary, N.C.,
USA).
[0094] Table V, below, shows the results of the simple logistic
regression analyses for describing the ability of each of the
parameters to distinguish individuals without coronary artery
disease from those with coronary artery disease.
5 TABLE V Area under the ROC- Parameter .chi..sup.2 df p-value
curve (AUC) Total cholesterol 115.06 4 0.0046 0.623 LDL 9.93 4
0.0416 0.591 HDL 26.49 2 <0.0001 0.671 Total chol/ 14.48 1
0.0001 0.630 HDL chol. Ratio Triglycerides 4.20 2 0.1227 0.576
Oxidized LDL 47.80 2 <0.0001 0.992 MDA-LDL 24.11 2 <0.0001
0.826
[0095] The area under the ROC-curve ("AUC") is 0.992 for OxLDL,
which is almost a perfect score (1 is the maximum AUC). This
indicates that the clinical presence of OxLDL above a predetermined
level can indicate with a very high degree of diagnostic accuracy
the presence of coronary artery disease as opposed to the absence
of CAD. In fact, the very high degree of diagnostic accuracy is
above the most preferred AUC minimum of 0.98. The only other AUC
value that is anywhere near that value for OxLDL is the AUC value
for MDA-modified LDL, which is 0.826, but even that is below the
minimum of 0.875 for a "very high degree of diagnostic accuracy."
All the other AUC values are substantially lower. For example, for
total cholesterol, which for the last decade or so has been the
classic marker for determining whether someone has or is at risk
for CAD, is only 0.623, which is a "rather low accuracy" (see Zweig
et al., "ROC Curve Analysis: An Example Showing The Relationships
Among Serum Lipid And Apolipoprotein Concentrations In Identifying
Patients With Coronary Artery Disease," Clin. Chem. 1992, 38(8):
1425-1428, citing Swets, "Measuring The Accuracy Of Diagnostic
Systems," Science 1988, 240: 1285-1293).
[0096] Table VI, below, shows the results of the simple logistic
regression analyses for describing the ability of each of the
parameters to distinguish between an acute stage of coronary artery
disease (i.e., either unstable angina or acute myocardial
infarction) and a non-acute stage.
6 TABLE VI Area under the ROC- Parameter .chi..sup.2 df p-value
curve (AUC) Total cholesterol 0.00 1 0.9464 0.520 LDL 0.12 1 0.7278
0.503 HDL 4.8 1 0.0285 0.570 Total chol/ 1.79 1 0.1815 0.555 HDL
chol. Ratio triglycerides 7.45 2 0.0241 0.618 Oxidized LDL 13.33 5
0.0098 0.672 MDA-LDL 18.66 3 0.0003 0.967 Troponin 24.42 2
<0.0001 0.848 C-reactive protein 19.93 2 <0.0001 0.710
D-dimer 5.32 1 0.0211 0.595
[0097] The area under the ROC-curve ("AUC") is 0.967 for
MDA-modified LDL, which is a very high score. This indicates that
the clinical presence of MDA-modified LDL above a predetermined
level can indicate with a very high degree of diagnostic accuracy
the presence of an acute stage of coronary artery disease (as
opposed to a non-acute stage). In fact, the very high degree of
diagnostic accuracy is above the preferred AUC minimum of 0.95. The
only other AUC value that is anywhere near that value for
MDA-modified LDL is the AUC value for troponin I, which is 0.848.
All the other AUC values are substantially lower.
[0098] Table VII, below, shows the results of the simple logistic
regression analyses for describing the ability of each of the
parameters to distinguish between unstable angina and acute
myocardial infarction.
7 TABLE VII Area under the ROC- Parameter .chi..sup.2 df p-value
curve (AUC) Total cholesterol 2.00 1 0.1572 0.598 LDL 0.11 1 0.7451
0.531 HDL 5.75 1 0.0165 0.625 Total chol/ 0.6 1 0.4371 0.587 HDL
chol. Ratio triglycerides 1.64 1 0.1997 0.539 Oxidized LDL 1.62 1
0.2028 0.579 MDA-LDL 1.66 1 0.1977 0.586 Troponin 22.26 2
<0.0001 0.777 C-reactive protein 5.24 2 0.0730 0.637 D-dimer
0.16 1 0.6892 0.568
[0099] The area under the ROC-curve ("AUC") is 0.777 for troponin
I, which, according to Swets (quoted in Zweig et al., Clin. Chem.
1992, 38(8): 1425-1428, above), indicates an accuracy useful for
some purposes. This AUC value of 0.777 indicates that the clinical
presence of troponin I above a predetermined level can indicate
with a high degree of diagnostic accuracy the presence of acute
myocardial infarction (as opposed to unstable angina). In fact, the
high degree of diagnostic accuracy is well above the preferred AUC
minimum of 0.70. The next highest AUC value is the AUC value for
C-reactive protein, which is 0.637. All the other AUC values,
including those for OxLDL and MDA-modified LDL are substantially
lower and are barely above the minimum AUC value of 0.5.
[0100] Table VIII, below, shows the results of the simple logistic
regression analyses for describing the relation between each of the
parameters and distinguishing between stable coronary artery
disease (either asymptomatic coronary artery disease or stable
angina) and unstable angina.
8 TABLE VIII Area under the ROC- Parameter .chi..sup.2 df p-value
curve (AUC) Total cholesterol 0.80 1 0.3709 0.534 LDL 0.26 1 0.6122
0.526 HDL 0.28 1 0.5998 0.507 Total chol/ 0.34 1 0.5618 0.514 HDL
chol. Ratio triglycerides 10.08 4 0.0391 0.701 Oxidized LDL 10.53 3
0.0415 0.689 MDA-LDL 24.56 1 <0.0001 0.997 Troponin 14.88 2
0.0006 0.743 C-reactive protein 9.05 2 0.0108 0.631 D-dimer 4.66 1
0.0308 0.641
[0101] The AUC for MDA-modified LDL is 0.997 (almost a perfect
value of 1), which shows that using MDA-modified LDL as marker can
distinguish with a very high degree of diagnostic accuracy between
stable coronary artery disease and unstable angina. No other
parameter comes close to matching the accuracy of MDA-modified
LDL.
[0102] Table IX, below, shows the results of the simple logistic
regression analyses for describing the relation between each of the
parameters and distinguishing between stable coronary artery
disease (either asymptomatic coronary artery disease or stable
angina) and acute myocardial infarction.
9 TABLE IX Area under the ROC- Parameter .chi..sup.2 df p-value
curve (AUC) Total cholesterol 0.58 1 0.4448 0.573 LDL 0.03 1 0.8742
0.488 HDL 7.27 1 0.0070 0.613 Total chol/ 2.56 1 0.1098 0.585 HDL
chol. Ratio triglycerides 5.49 2 0.0644 0.617 Oxidized LDL 4.21 1
0.0401 0.585 MDA-LDL 23.74 2 <0.0001 0.967 Troponin 28.75 2
<0.0001 0.921 C-reactive protein 25.60 2 <0.0001 0.763
D-dimer 4.19 1 0.0406 0.562
[0103] The AUC for MDA-modified LDL is 0.967, which shows that
using MDA-modified LDL as marker can distinguish with a very high
degree of diagnostic accuracy between stable coronary artery
disease and acute myocardial infarction. Troponin I, with an AUC
value of 0.921 is good but not nearly as perfect. The next highest
AUC value is 0.763, for C-reactive protein, but that is
significantly lower than the MDA-modified LDL and troponin I AUC
values.
[0104] All of these results show that the present invention
provides a method having a clinically sufficient degree of
diagnostic accuracy for detecting the presence of and for
distinguishing between or among the non-acute and the acute stages
of coronary artery disease for a human patient from the general
population, the non-acute stage of coronary artery disease being
either asymptomatic coronary artery disease or stable angina and
the acute stages of coronary artery disease being unstable angina
and acute myocardial infarction.
[0105] Variations and modifications will be apparent to those
skilled in the art, and the claims are intended to cover all
variations and modifications that fall within the true spirit and
scope of the invention.
[0106] For example, the cut-points for the various markers will
depend on which markers are used and which tests are used. When
using the methods described herein, the cut-point may be 1.4 mg/dL
(milligrams/deciliter) for OxLDL, 0.7 mg/dL for MDA-modified LDL,
and 0.07 ng/mL (nanograms/milliliter) for troponin I. However, if,
for example, a non-ionic detergent such as Tween 20
(polyoxyethylenesorbitan monolaurate; Sigma Chemical Company) is
included in the buffer solution (the PBS solution) with which the
LDL-containing material (e.g., plasma, standard, or control) is
incubated (e.g., in a concentration in the buffer solution of up to
about 1% w/v [weight/volume], with a value in the range of about
0.2% w/v to about 0.6% w/v appearing to be optimum), the OxLDL and
MDA-modified LDL values may be significantly increased, in which
case the respective cut-points would have to be increased. Without
wishing to be bound by any theory, it is believed that a non-ionic
detergent will separate the protein portion from the lipid portion
of the OxLDL and MDA-modified LDL, that the preferred monoclonal
antibodies mAb-4E6 and mAb-1H11 are directed to epitopes on the
protein portion, and that removing the lipid portion from the
protein portion removes steric hindrance and allows the antibody to
bind to more sites on the same protein portion, thereby increasing
the total amount of antibody that binds to a given amount of OxLDL
or MDA-modified LDL. Thus, it has been observed that use of Tween
20 in a concentration of 0.2% w/v to 0.6% w/v in the buffer with a
freshly drawn plasma sample increased the reported amount of OxLDL
in the sample by a factor of over 1 0-fold as compared to when no
Tween 20 was used in the buffer for the same sample amount of the
same plasma. That is desirable because, broadly speaking, having a
larger range for a marker whose presence above a predetermined
value in a test indicates the presence of a disease, condition, or
syndrome can increase the accuracy of the test.
* * * * *