ASSAY FOR DIAGNOSIS OF CARDIAC MYOCYTE DAMAGE

• US 20110129854A1
• Filed: 12/03/2009
• Published: 06/02/2011
• Est. Priority Date: 12/02/2009
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Adamczyk, Maciej, Mattingly, Phillip G., Brashear, Jeffrey R.
• Granted Patent: US 8,652,788 B2
• Time in Patent Office: Days
• US Class Current: 435/7.21
• CPC Class Codes: C07D 219/14 with hydrocarbon radicals, substituted by nitrogen atoms, attached to the ring nitrogen atom
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 2333/5412 IL-6
• CPC Class Codes: G01N 2333/58 Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
• CPC Class Codes: G01N 2800/325 Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/6878 in eptitope analysis
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

Assays are disclosed for diagnosing a clinical condition, assessing risk or predicting an outcome as a result of cardiac myocyte damage. Immunoassay methods and kits provide for the assessment of cardiac myocyte damage by determining the presence of multiple cardiac myocyte antigens in a test sample, and combining the multiple determinations in a single assay result.

Claims

1. An immunoassay for detecting cardiac myocyte damage in a subject from a test sample, the immunoassay comprising the steps of:

a) contacting a test sample from a subject suspected of having cardiac myocyte damage with n antibodies (A_an′

ⁿ) that bind to at least n epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

(n-antibody;

antigen) immuno-complexes (A_a1¹)(A_a1²) . . . (A_a1ⁿ)(a¹), (A_a1²)(A_a2²) . . . (A_a2ⁿ)(a²), . . . (A_an′

¹)(A_an′

²) . . . (A_an′

ⁿ)(a^n′

) wherein n is an integer from 1 to 10; and

n′

is an integer from 2 to 10;

b) contacting said mixture comprising n′

(n-antibody;

antigen) immuno-complexes with n″

antibodies (B_an′

^n″

) that bind to n″

epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

((n+n″

) antibody;

antigen) measurable assemblies ((A_a1¹)(A_a1²) . . . (A_a1ⁿ)((B_a1¹)(B_a1²) . . . (B_a1ⁿ)(a¹), ((A_a2¹)(A_a2²) . . . (A_a2ⁿ))((B_a2¹)(B_a2²) . . . (A_a2ⁿ))(a²), . . . ((A_an′

¹)(A_an′

²) . . . (A_an′

ⁿ)(B_an″

¹)(B_an′

²) . . . (B_an″

ⁿ))(a^n′

) wherein n and n″

are independently an integer from 1 to 10, and n′

is an integer from 2 to 10, and antibodies A and B bind to (n+n″

) different epitopes of a cardiac myocyte antigen;

c) measuring an optical, electrical, or change-of-state signal of the measurable assembly; and

d) detecting cardiac myocyte damage by determining whether the measurement in step (c) exceeds a predetermined level.

2. The immunoassay of claim 1, wherein the cardiac myocyte antigens a^n′

are selected from the group consisting of cardiac troponin-I, cardiac troponin-T, creatine phosphokinase MB (CKMB), myoglobin, myosin heavy chain, myosin light chain, B-type natriuretic peptide (including pro-BNP, NT-proBNP, and hBNP(1-32)), heart fatty-acid-binding protein (H-FABP), placenta growth factor (PLGF), and interleukin-6 (IL-6).

3. The immunoassay of claim 1, wherein n′

=2 and the cardiac myocyte antigens a^n′

are cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT).

4. The immunoassay of claim 1 or 2, wherein the antibodies A_an′

ⁿ, antibodies B_an′

^n″

or antibodies A_an′

ⁿ and B_an′

^n″

comprise humanized antibodies.

5. The immunoassay of claim 1 or 2, wherein the antibodies B_an′

^n″

comprise anti-human IgG antibodies.

6. The immunoassay of claim 1 or 2, wherein the antibodies B_an′

^n″

are bound to a detectable label, wherein the detectable label is an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin.

7. The immunoassay of claim 1 or 2, wherein the optical signal is measured as a cardiac myocyte antigens a^n′

concentration dependent change in chemiluminescence, fluorescence, phosphorescence, electrochemiluminescence, ultraviolet absorption, visible absorption, infrared absorption, refraction, surface plasmon resonance.

8. The immunoassay of claim 1 or 2, wherein the electrical signal is measured as a cardiac myocyte antigens a^n′

concentration dependent change in current, resistance, potential, mass to charge ratio, or ion count.

9. The immunoassay of claim 1 or 2, wherein the change-of-state signal is measured as a cardiac myocyte antigens a^n′

concentration dependent change in size, solubility, mass, or resonance.

10. The immunoassay of claim 1 or 2, wherein the antibodies A_an′

ⁿ are immobilized on a solid phase.

11. The immunoassay of claim 10, wherein the solid phase is selected from the group consisting of a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, a scaffolding molecule, quartz crystal, film, filter paper, disc and chip.

12. The immunoassay of claim 1 or 2, wherein the test sample is whole blood, serum, or plasma.

13. An immunoassay for detecting cardiac myocyte damage in a subject from a test sample, the immunoassay comprising the steps of:

a) contacting a test sample from a subject suspected of having cardiac myocyte damage with n antibodies (A_an′

ⁿ) that bind to at least n epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

(n-antibody;

antigen) immuno-complexes (A_a1¹)(A_a1²) . . . (A_a1ⁿ)(a¹), (A_a2¹)(A_a2²) . . . (A_a1ⁿ)(a²), . . . (A_an′

¹)(A_an′

²) . . . (A_an′

ⁿ)(a^n′

) wherein n is an integer from 1 to 10; and

n′

is an integer from 2 to 10;

b) contacting said mixture comprising n′

(n-antibody;

antigen) immuno-complexes with n″

antibodies (B_an′

^n″

) that bind to n″

epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

((n+n″

) antibody;

antigen) measurable assemblies ((A_a1¹)(A_a1²) . . . (A_a1ⁿ)((B_a1¹)(B_a1²) . . . (B_a1ⁿ)(a¹), ((A_a2¹)(A_a2²) . . . (A_a2ⁿ))((B_a2¹)(B_a2²) . . . (B_a2ⁿ))(a²), . . . ((A_an′

¹)(A_an′

²) . . . (A_an′

ⁿ))((B_an″

¹)(B_an″

²) . . . (B_an″

ⁿ)(a^n′

) wherein n and n″

are independently an integer from 1 to 10, and n′

is an integer from 2 to 10, and antibodies A and B bind to (n+n″

) different epitopes of a cardiac myocyte antigen, wherein antibodies B are labeled with a detectable label comprising at least one acridinium compound;

c) generating or providing a source of hydrogen peroxide to the mixture of step (b);

d) adding a basic solution to the mixture of step (c) to generate a light signal;

e) measuring the light signal generated by or emitted in step (d); and

f) detecting cardiac myocyte damage by determining whether the measurement in step (e) exceeds a predetermined level.

14. The immunoassay of claim 13, wherein the cardiac myocyte antigens a^n′

are selected from the group consisting of cardiac troponin-I, cardiac troponin-T, creatine phosphokinase MB (CKMB), myoglobin, myosin heavy chain, myosin light chain, B-type natriuretic peptide (including pro-BNP, NT-proBNP, and hBNP(1-32)), heart fatty-acid-binding protein (H-FABP), placenta growth factor (PLGF), and interleukin-6 (IL-6).

15. The immunoassay of claim 13, wherein n′

=2 and the cardiac myocyte antigens a^n′

are cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT).

16. The immunoassay of claim 13 or 14, wherein the antibodies A_an′

ⁿ, antibodies B_an′

^n″

, or antibodies A_an′

ⁿ and B_a1^n″

comprise humanized antibodies.

17. The immunoassay of claim 13 or 14, wherein the antibodies B_an′

^n″

comprise anti-human IgG antibodies.

18. The immunoassay of claim 13 or 14, wherein the antibodies A_an′

ⁿ are immobilized on a solid phase.

19. The immunoassay of claim 18, wherein the solid phase is selected from the group consisting of a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, a scaffolding molecule, quartz crystal, film, filter paper, disc and chip.

20. The immunoassay of claim 13 or 14, wherein the test sample is whole blood, serum, or plasma.

21. The immunoassay of claim 13 or 14, wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I:

22. The immunoassay of claim 13 or 14, wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

23. The immunoassay of claim 13 or 14, wherein the antibodies A_an′

ⁿ are selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody.

24. The immunoassay of claim 13 or 14, wherein the antibodies B_an′

^n″

are selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody.

25. The immunoassay of claim 13 or 14, wherein the hydrogen peroxide is provided by adding a buffer or a solution containing hydrogen peroxide.

26. The immunoassay of claim 13 or 14, wherein the hydrogen peroxide is generated by adding a hydrogen peroxide generating enzyme to the test sample.

27. The immunoassay of claim 26, wherein the hydrogen peroxide generating enzyme is selected from the group consisting of:

(R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acid oxidase, (S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase, 3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase, 6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoA oxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase (copper-containing), amine oxidase (flavin-containing), aryl-alcohol oxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase, choline oxidase, columbamine oxidase, cyclohexylamine oxidase, cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactone oxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase, D-glutamate oxidase, D-glutamate(D-aspartate) oxidase, dihydrobenzophenanthridine oxidase, dihydroorotate oxidase, dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase, ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucose oxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycine oxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase, indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acid oxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamate oxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine oxidase, long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase, malate oxidase, methanethiol oxidase, monoamino acid oxidase, N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase, nitroalkane oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxidase, oxalate oxidase, polyamine oxidase, polyphenol oxidase, polyvinyl-alcohol oxidase, prenylcysteine oxidase, protein-lysine 6-oxidase, putrescine oxidase, pyranose oxidase, pyridoxal 5′

-phosphate synthase, pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase, pyruvate oxidase, pyruvate oxidase (CoA-acetylating), reticuline oxidase, retinal oxidase, rifamycin-B oxidase, sarcosine oxidase, secondary-alcohol oxidase, sulfite oxidase, superoxide dismutase, superoxide reductase, tetrahydroberberine oxidase, thiamine oxidase, tryptophan α

,β

-oxidase, urate oxidase (uricase, uric acid oxidase), vanillyl-alcohol oxidase, xanthine oxidase, xylitol oxidase and combinations thereof.

28. The immunoassay of claim 13 or 14, wherein the basic solution is a solution having a pH of at least about 10.

29. The immunoassay of claim 13 or 14, wherein step f) detecting cardiac myocyte damage by determining whether the measurement in step (e) exceeds a predetermined level comprises relating the amount of light signal in step (e) to the amount of cardiac antigens a and b in the test sample either by use of a standard curve for each cardiac antigen a or by comparison to a reference standard for each cardiac antigen a.

30. The immunoassay of claim 29, comprising comparison to a reference standard for each cardiac antigen a^n′

, each reference standard comprising an anti-idiotypic antibody.

31. The immunoassay of claim 29, comprising comparison to a reference standard for each cardiac antigen a^n′

, each reference standard comprising a derivatized cardiac myocyte antigen.

32. The immunoassay of claim 31, wherein the derivatized cardiac myocyte antigen comprises the cardiac myocyte antigen derivatized with a polyethylene glycol.

33. The immunoassay of claim 13 or 14, wherein the immunoassay is adapted for use in an automated system or semi-automated system.

34. A kit for detecting cardiac myocyte damage from a test sample, the kit comprising:

a) n antibodies (A_an′

ⁿ) that bind to at least n epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

(n-antibody;

antigen) immuno-complexes (A_a1¹)(A_a1²) . . . (A_a1ⁿ)(a¹), (A_a2¹)(A_a2²) . . . (A_a2ⁿ)(a²), . . . (A_an′

¹)(A_an²) . . . (A_an′

ⁿ)(a^n′

) wherein n is an integer from 1 to 10; and

n′

is an integer from 2 to 10;

b) n″

antibodies (B_an′

^n″

) that bind to n″

epitopes on n′

cardiac myocyte antigens (a^n′

) to form n′

((n+n″

) antibody;

antigen) measurable assemblies ((A_a1¹)(A_a1²) . . . (A_a1ⁿ))((B_a1¹)(B_a1²) . . . (B_a1ⁿ))(a¹), ((A_a2¹)(A_a2²) . . . (A_a2ⁿ))((B_a2¹)(B_a2²) . . . (B_a2ⁿ)(a²), . . . ((A_an′

¹)(A_an′

²) . . . (A_an′

ⁿ))((B_an″

¹)(B_an″

²) . . . (B_an″

ⁿ))(a^n′

), wherein n and n″

are independently an integer from 1 to 10, and n′

is an integer from 2 to 10, and antibodies A and B bind to (n+n″

) different epitopes of a cardiac myocyte antigen; and

c) instructions for determining whether the total amount of cardiac myocyte antigens (a^n′

) in the test sample exceeds a predetermined

35. The kit of claim 34, wherein the cardiac myocyte antigens an are selected from the group consisting of cardiac troponin-I, cardiac troponin-T, creatine phosphokinase MB (CKMB), myoglobin, myosin heavy chain, myosin light chain, B-type natriuretic peptide, wherein the first cardiac specific antigen and the second cardiac specific antigen are different antigens.

36. The kit of claim 34, wherein n′

=2 and the cardiac myocyte antigens a^n′

are cardiac troponin-I (cTnI) and cardiac troponin-T cTnT.

37. The kit of claim 34 or 35, further comprising a solid phase wherein the antibodies A_an′

ⁿ are bound to the solid phase.

38. The kit of claim 37, wherein the solid phase is selected from the group consisting of a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal, a film, a filter paper, a disc and a chip.

39. The kit of claim 34 or 35, further comprising a detectable label bound to the antibodies B_an′

^n″

.

40. The kit of claim 39, wherein the detectable label is an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin.

41. The kit of claim 40, wherein the detectable label is an acridinium compound.

42. The kit of claim 41, wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I:

43. The kit of claim 41, wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

44. The kit of claim 42 or 43, further comprising a basic solution.

45. The kit of claim 44, wherein the basic solution is a solution having a pH of at least about 10.

46. The kit of claim 45, further comprising a hydrogen peroxide source.

47. The kit of claim 46, wherein the hydrogen peroxide source comprises a buffer or a solution containing hydrogen peroxide.

48. The kit of claim 47, wherein the hydrogen peroxide source comprises a hydrogen peroxide generating enzyme.

49. The kit of claim 48, wherein the hydrogen peroxide generating enzyme is selected from the group consisting of:

(R)-6-hydroxynicotine oxidase, (S)-2-hydroxy acid oxidase, (S)-6-hydroxynicotine oxidase, 3-aci-nitropropanoate oxidase, 3-hydroxyanthranilate oxidase, 4-hydroxymandelate oxidase, 6-hydroxynicotinate dehydrogenase, abscisic-aldehyde oxidase, acyl-CoA oxidase, alcohol oxidase, aldehyde oxidase, amine oxidase, amine oxidase (copper-containing), amine oxidase (flavin-containing), aryl-alcohol oxidase, aryl-aldehyde oxidase, catechol oxidase, cholesterol oxidase, choline oxidase, columbamine oxidase, cyclohexylamine oxidase, cytochrome c oxidase, D-amino-acid oxidase, D-arabinono-1,4-lactone oxidase, D-arabinono-1,4-lactone oxidase, D-aspartate oxidase, D-glutamate oxidase, D-glutamate(D-aspartate) oxidase, dihydrobenzophenanthridine oxidase, dihydroorotate oxidase, dihydrouracil oxidase, dimethylglycine oxidase, D-mannitol oxidase, ecdysone oxidase, ethanolamine oxidase, galactose oxidase, glucose oxidase, glutathione oxidase, glycerol-3-phosphate oxidase, glycine oxidase, glyoxylate oxidase, hexose oxidase, hydroxyphytanate oxidase, indole-3-acetaldehyde oxidase, lactic acid oxidase, L-amino-acid oxidase, L-aspartate oxidase, L-galactonolactone oxidase, L-glutamate oxidase, L-gulonolactone oxidase, L-lysine 6-oxidase, L-lysine oxidase, long-chain-alcohol oxidase, L-pipecolate oxidase, L-sorbose oxidase, malate oxidase, methanethiol oxidase, monoamino acid oxidase, N6-methyl-lysine oxidase, N-acylhexosamine oxidase, NAD(P)H oxidase, nitroalkane oxidase, N-methyl-L-amino-acid oxidase, nucleoside oxidase, oxalate oxidase, polyamine oxidase, polyphenol oxidase, polyvinyl-alcohol oxidase, prenylcysteine oxidase, protein-lysine 6-oxidase, putrescine oxidase, pyranose oxidase, pyridoxal 5′

-phosphate synthase, pyridoxine 4-oxidase, pyrroloquinoline-quinone synthase, pyruvate oxidase, pyruvate oxidase (CoA-acetylating), reticuline oxidase, retinal oxidase, rifamycin-B oxidase, sarcosine oxidase, secondary-alcohol oxidase, sulfite oxidase, superoxide dismutase, superoxide reductase, tetrahydroberberine oxidase, thiamine oxidase, tryptophan α

,β

-oxidase, urate oxidase (uricase, uric acid oxidase), vanillyl-alcohol oxidase, xanthine oxidase, xylitol oxidase and combinations thereof.

50. The kit of claim 34 or 35, wherein the detection antibodies comprise anti-human IgG antibodies.

51. The kit of claim 34 or 35, further comprising at least one cardiac myocyte antigen reference standard.

52. The kit of claim 51, wherein the at least one cardiac myocyte antigen reference standard comprises an anti-idiotypic antibody.

53. The kit of claim 51, wherein the at least one cardiac myocyte antigen reference standard comprises a derivatized cardiac myocyte antigen.

54. The kit of claim 53, wherein the derivatized cardiac myocyte antigen comprises the cardiac myocyte antigen derivatized with a polyethylene glycol.