Methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays

• US 6,174,686 B1
• Filed: 04/18/1996
• Issued: 01/16/2001
• Est. Priority Date: 04/18/1995
• Status: Expired due to Term

• Current Assignee: BIOSITE INCORPORATED (Abbott Laboratories)
• Original Assignee: Biosite Diagnostics Incorporated (Abbott Laboratories)
• Inventors: Buechler, Kenneth F., McPherson, Paul H.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): Nguyen, Bao-Thuy L.
• Application Number:
  US08/633,248
• Time in Patent Office: 1,734 Days
• Field of Search: 435/7.1, 435/7.2, 435/7.5, 435/7.71, 435/7.72, 435/7.8-7.95, 435/960, 435/962, 435/971, 435/973, 436/518, 436/523, 436/528, 436/535, 436/538, 436/811, 436/819
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 2800/32 Cardiovascular disorders
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody

Abstract

Assay systems and specialized antibodies for the detection and quantitation of troponin I and troponin T in body fluids as an indicator of myocardial infarction. Since troponin I and T exist in various conformations in the blood, the ratios of the monomeric troponin I an T and the binary and ternary complexes, as well as which form of troponin present in the blood, may be related to the metabolic state of the heart. Disclosed is a system to determine the presence of a troponin form or a group of troponin forms in a sample of whole blood, serum or plasma.

Disclosed is a stabilized composition of troponin; the stabilized composition can comprise a stabilized composition of troponin I, wherein the troponin I is oxidized, the troponin I can be unbound or the troponin I can be in a complex.

Disclosed is a method for improving the recovery of troponin I or T from a surface used in immunoassays

Also disclosed are antibodies which recognize, unbound troponin forms, the forms of troponin in binary complexes, the ternary complex of troponin I, T and C, and the conformations of troponin I having intramolecularly oxidized and reduced cysteines.

Claims

1. An assay for determining the presence or amount of all free cardiac specific troponin isoforms and complexed isoforms of cardiac troponin in a blood sample, said complexed isoforms comprising isoforms selected from the group consisting of troponin I, troponin C and troponin T, wherein said assay comprises:

contacting said sample with at least one inhibitor, wherein said inhibitor causes dissociation of said complexed isoforms; and

performing an immunoassay with antibody specific to cardiac troponin, wherein said antibody binds said free isoforms in said sample,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin isoforms in said sample.

2. The assay of claim 1, wherein said antibody comprises a plurality of antibodies selected from the group consisting of recombinant and monoclonal antibodies.

7. The assay of any one of claims 1 and 3-6, wherein said inhibitor is melittin.

8. The assay of any one of claims 1 and 3-6 wherein said inhibitor is mastoparan.

9. The assay of any one of claims 1 and 3-6 comprising at least two inhibitors, one of which is a metal chelator and another of which is selected from the group consisting of mastoparan and melittin.

22. The assay of any one of claims 1, 3-6, 13, and 18-21, wherein said assay provides a method for diagnosing a myocardial infarction, wherein said method comprises correlating the results of said assay with the existence of free and complexed troponin in said sample.

3. An assay for determining the presence or amount of a free and complexed specific isoform of cardiac troponin in a blood sample, said complexed isoform comprising isoforms selected from the group consisting of troponin I, troponin C and troponin T, wherein said assay comprises:

contacting said sample with at least one inhibitor, wherein said inhibitor causes dissociation of said complexed isoform; and

performing an immunoassay with antibody specific to cardiac troponin, wherein said antibody binds said isoform in said sample,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin isoform in said sample.

4. An assay for determining the presence or amount of a free and complexed specific isoform of cardiac troponin in a blood sample, said complexed isoform comprising isoforms selected from the group consisting of troponin I, troponin C and troponin T, wherein said assay comprises:

contacting said sample with at least one inhibitor, wherein said inhibitor causes dissociation of said complexed isoform; and

performing an immunoassay with antibody specific to cardiac troponin I and/or T, wherein said antibody binds said isoform in said sample,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin isoform in said sample.

5. An assay for determining the presence or amount of a free and complexed cardiac specific troponin I in a blood sample, said complexed cardiac specific troponin I comprising isoforms selected from the group consisting of troponin C and troponin T, wherein said assay comprises:

contacting said sample with at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin I; and

performing an immunoassay with antibody specific to cardiac troponin I, wherein said antibody binds said cardiac specific troponin I in said sample,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin I in said sample.

6. An assay for determining the presence or amount of free and complexed cardiac specific troponin T in a blood sample, said complexed cardiac specific troponin T comprising at least one isoform selected from the group consisting of troponin I and troponin C, wherein said assay comprises:

contacting said sample with at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin T; and

performing an immunoassay with antibody specific to cardiac troponin T, wherein said antibody binds said cardiac specific troponin T in said sample,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin T in said sample.

10. An assay for determining the presence or amount of a cardiac specific complexed isoform of troponin in a blood sample, wherein said cardiac specific complexed isoform of troponin is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, wherein said assay comprises:

performing an immunoassay with at least one antibody conjugate which binds at least one of said complexes in said sample, and at least one capture antibody which binds to said antibody conjugate,

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said complexed cardiac specific troponin isoform in said sample.

17. The assay of any one of claims 10, 11, 12, and 14, wherein said assay provides a method for diagnosing a myocardial infarction, wherein said method comprises correlating the results of said assay with the existence of complexed troponin in said sample.

11. An immunoassay method for determining the presence or amount of complexes of a cardiac specific isoform of troponin in a blood sample, said method comprising:

a) providing an antibody conjugate comprising an antibody which specifically binds to the cardiac specific isoform of troponin in a complex comprising said isoform and another troponin component, and a signal generating element;

or,

providing an antibody conjugate comprising an antibody which specifically binds to the cardiac specific isoform of troponin in a binary complex comprising said isoform and another troponin component, and a signal generating element; and

, providing an antibody conjugate comprising an antibody which specifically binds to the cardiac specific isoform of troponin in a ternary complex of three troponin components, and a signal generating element;

b) contacting said sample with said antibody conjugate(s) of step a), whereby the antibody conjugate(s) bind to said troponin complexes present in the sample;

c) contacting the sample with a solid phase to which is bound at least one capture antibody which specifically binds to said antibody conjugate(s) bound to said troponin complexes, whereby a detectable signal is present from said antibody conjugate(s) upon binding to the capture antibody; and

,

d) relating the detectable signal to the presence or amount of complexes of the cardiac specific isoform of troponin in said sample.

12. An immunoassay method for determining the presence or amount of complexes comprising any cardiac specific isoform of troponin in a blood sample, said method comprising:

a) providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in complexes comprising two or more troponin components, and a signal generating element;

or,

providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in binary complexes comprising two troponin components, and a signal generating element; and

, providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in ternary complexes comprising three troponin components, and a signal generating element; and

,

b) contacting said sample with said antibody conjugate(s) of step a), whereby the antibody conjugate(s) bind to troponin complexes present in the sample;

c) contacting the sample with a solid phase to which is bound at least one capture antibody which specifically binds said antibody conjugate(s) bound to said troponin complexes, whereby a detectable signal is present from the antibody conjugate(s) upon binding to the capture antibody; and

,

d) relating the detectable signal to the presence or amount of the cardiac specific isoform of troponin in said sample.

13. An assay for determining the presence or amount of all free and complexed cardiac specific troponin isoforms in a blood sample, wherein said complexed cardiac specific troponin isoforms are in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, the assay comprising:

performing an immunoassay with at least one antibody conjugate, the antibody conjugate binding said free and complexed cardiac specific troponin isoforms in said sample, and at least one capture antibody which specifically binds to said antibody conjugate; and

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and complexed cardiac specific troponin isoforms in said sample.

15. The assay of either one of claims 13 or 14, wherein said immunoassay provides a quantitative assay for each of said isoforms.

16. The assay of any one of claims 13, or 14, wherein said at least one antibody conjugate and said at least one capture antibody are selected from the group consisting of recombinant antibody, polyclonal antibody and monoclonal antibody.

14. An assay for determining the presence or amount of complexed cardiac specific troponin isoforms in a blood sample, wherein said complexed cardiac specific isoforms are in one or more complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, the assay comprising:

performing an immunoassay with at least one antibody conjugate, the antibody conjugate binding said complexed cardiac specific troponin isoforms in said sample, and at least one capture antibody which binds to said antibody conjugate; and

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said complexed cardiac specific isoforms in said blood sample.

18. An assay for determining the presence or amount of a free and complexed cardiac specific isoform of troponin in a blood sample, wherein said complexed cardiac specific isoform of troponin is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, the assay comprising:

performing an immunoassay with at least one antibody conjugate, the antibody conjugate binding said free and complexed cardiac specific isoform of troponin in said sample, and at least one capture antibody which binds to said antibody conjugate; and

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and complexed cardiac specific troponin isoform in said sample.

19. An assay for determining the presence or amount of a free and complexed cardiac specific isoform of troponin in a blood sample, wherein said complexed cardiac specific isoform of troponin is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, the assay comprising:

performing an immunoassay with at least one antibody conjugate specific to cardiac troponin I and/or T, the antibody conjugate binding said free and complexed cardiac specific isoform of troponin in said sample, and at least one capture antibody which binds to said antibody conjugate; and

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and complexed cardiac specific troponin isoform in said sample.

20. An assay for determining the presence or amount of free and complexed cardiac specific troponin I in a blood sample, wherein said complexed cardiac specific troponin I is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin C and troponin T, the assay comprising:

performing an immunoassay with at least one antibody conjugate specific to cardiac troponin I, the antibody conjugate binding said free and complexed cardiac specific troponin I in said sample, and at least one capture antibody which binds to said antibody conjugate; and

wherein a detectable signal of said immunoassay is related to the presence or amount of said free and complexed cardiac specific troponin I in said sample.

21. An assay for determining the presence or amount of free and complexed cardiac specific troponin T in a blood sample, wherein said complexed cardiac specific troponin T is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, and troponin C, the assay comprising:

performing an immunoassay with at least one antibody conjugate specific to cardiac troponin T, the antibody conjugate binding said free and complexed cardiac specific troponin T in said sample, and at least one capture antibody which binds to said antibody conjugate; and

wherein a detectable signal derived from said immunoassay is related to the presence or amount of said free and said complexed cardiac specific troponin T in said sample.

23. A kit for determining the presence or amount of all free and complexed cardiac specific troponin isoforms in a blood sample, wherein said complexed cardiac specific troponin isoforms are in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, wherein said kit comprises:

at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin isoforms; and

antibody specific to cardiac troponin, wherein said antibody binds said free cardiac specific troponin isoforms in said sample.

24. The kit of claim 23, wherein said antibody comprises a plurality of monoclonal antibodies.

29. The kit of any one of claims 23 and 25-28, wherein said inhibitor is mellitin.

30. The kit of any one of claims 23 and 25-28, wherein said inhibitor is mastoparan.

31. The kit of any one of claims 23 and 25-28 comprising at least two inhibitors, one of which is a metal chelator and another of which is selected from the group consisting of mastoparan and melittin.

25. A kit for determining the presence or amount of a free and complexed specific isoform of cardiac troponin in a blood sample, wherein said complexed cardiac specific troponin isoform is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, wherein said kit comprises:

at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin isoform; and

antibody specific to cardiac troponin, wherein said antibody binds said free cardiac specific troponin isoform in said sample.

26. A kit for determining the presence or amount of a free and complexed cardiac specific isoform of troponin in a blood sample, wherein said complexed cardiac specific troponin isoform is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, troponin C and troponin T, wherein said kit comprises:

at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin isoform; and

antibody specific to cardiac troponin I and/or T, wherein said antibody binds said cardiac specific troponin isoform in said sample.

27. A kit for determining the presence or amount of free and complexed cardiac specific troponin I in a blood sample, wherein said complexed cardiac specific troponin I is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin C and troponin T, wherein said kit comprises:

at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin I; and

antibody specific to cardiac troponin I, wherein said antibody binds said cardiac specific troponin I in said sample.

28. A kit for determining the presence or amount of free and complexed cardiac specific troponin T in a blood sample, wherein said complexed cardiac specific troponin T is in one or more complexes, said complexes comprising at least one other troponin component selected from the group consisting of troponin I, and troponin C, wherein said kit comprises:

at least one inhibitor, wherein said inhibitor causes dissociation of said complexed cardiac specific troponin T; and

antibody specific to cardiac troponin T, wherein said antibody binds said cardiac specific troponin T in said sample.

32. An assay for determining the presence or amount of at least one free and complexed cardiac specific troponin isoform in a blood sample, said complexed troponin isoform comprising isoforms selected from the group consisting of troponin I, troponin C and troponin T, wherein said assay comprises:

performing an immunoassay with antibody which specifically binds said at least one free and complexed cardiac specific troponin isoform;

wherein the results of said assay indicate the total amount of said at least one free and complexed cardiac specific troponin isoform; and

,

wherein said assay provides a method for diagnosing a myocardial infarction, wherein said method comprises correlating the results of said assay with the existence of free and complexed troponin in said sample.

33. An immunoassay method for determining the presence or amount of complexes comprising any cardiac specific isoform of troponin in a blood sample, said method comprising:

a) providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in complexes comprising two or more troponin components, and a signal generating element;

or,

providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in binary complexes comprising two troponin components, and a signal generating element; and

, providing an antibody conjugate comprising an antibody which specifically binds to a cardiac specific isoform of troponin in ternary complexes comprising three troponin components, and a signal generating element; and

,

b) contacting said sample with said antibody conjugate of step a), whereby the antibody conjugates bind to troponin complexes present in the sample;

c) contacting the sample with a solid phase to which is bound at least one capture antibody which specifically binds to said antibody conjugate, whereby a detectable signal is present from the antibody conjugate upon binding to the capture antibody; and

,

d) relating the detectable signal to the presence or amount of the cardiac specific isoform of troponin in said sample.