ASSAY FOR CARDIAC TROPONIN-T (cTnT)

• US 20110129818A1
• Filed: 12/02/2009
• Published: 06/02/2011
• Est. Priority Date: 12/02/2009
• Status: Active Grant

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Brashear, Jeffrey R., Adamczyk, Maciej, Mattingly, Phillip G.
• Granted Patent: US 8,835,120 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 2800/32 Cardiovascular disorders
• CPC Class Codes: G01N 2800/324 Coronary artery diseases, e.g. angina pectoris, myocardial infarction
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present disclosure describes immunoassays for detecting cardiac troponin-T (cTnT) in a test sample, and in particular immunoassays and kits for detecting cTnT in a test sample suspected of containing substances that may interfere with the determination of cTnT, such as heterophilic endogenous antibodies and autoantibodies to cTnT. The methods use more than one capture phase antibody and more than one detection antibody to improve specificity, and provide for the use of humanized immunoreagents to overcome heterophilic antibody interferences.

Claims

1. An immunoassay for quantifying an amount of cardiac troponin-T (cTnT) in a test sample, the immunoassay comprising the steps of:

a) contacting a test sample suspected of containing cTnT with n capture antibodies (C) that bind to at least n epitopes on cTnT to form a n-capture antibody;

cTnT complex (C₁)(C₂) . . . (Cn)(cTnT), wherein n is an integer from 1 to 10; and

b) contacting said mixture comprising a n-capture antibody-cTnT epitope complex (C₁)(C₂) . . . (Cn)(cTnT) with n′

detection antibodies (D) that bind to n′

epitopes on cTnT to form a n′

-detection antibody;

n-capture antibody;

cTnT measurable assembly (D₁)(D₂) . . . (Dn′

) (C₁)(C₂) . . . (Cn)(cTnT); and

wherein n′ and

n are independently an integer from 1 to 10, and antibodies C and D bind to (n+n′

) different epitopes of cTnT.

2. The immunoassay of claim 1, wherein, an optical, electrical, or change-of-state signal of the measurable assembly is measured.

3. The immunoassay of claim 1 or 2, wherein the detection antibodies are conjugated to a detectable label, wherein the detectable label is an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin.

4. The immunoassay of claim 1 or 2, wherein the capture antibodies, the detection antibodies or capture and detection antibodies comprise humanized antibodies.

5. The immunoassay of claim 1 or 2, wherein the detection antibodies comprise humanized antibodies complexed with an anti-human IgG antibody.

6. The immunoassay of claim 5, wherein said anti-human IgG antibody is conjugated 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 2, wherein the optical signal is measured as a cTnT concentration dependent change in chemiluminescence, fluorescence, phosphorescence, electrochemiluminescence, ultraviolet absorption, visible absorption, infrared absorption, refraction, surface plasmon resonance.

8. The immunoassay of claim 2, wherein the electrical signal is measured as a troponin-T concentration dependent change in current, resistance, potential, mass to charge ratio, or ion count.

9. The immunoassay of claim 2, wherein the change-of-state signal is measured as a troponin-T concentration dependent change in size, solubility, mass, or resonance.

10. The immunoassay of claim 1 or 2, wherein the capture antibodies 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. An immunoassay for quantifying an amount of cardiac troponin-T (cTnT) in a test sample, the immunoassay comprising the steps of:

a) contacting a test sample suspected of containing cTnT with n capture antibodies (C) that bind to at least n epitopes on cTnT to form a n-capture antibody;

cTnT complex (C₁)(C₂) . . . (Cn)(cTnT), wherein n is an integer from 1 to 10;

b) contacting said mixture comprising a n-capture antibody-cTnT epitope complex (C₁)(C₂) . . . (Cn)(cTnT) with n′

detection antibodies (D) that bind to n′

epitopes on cTnT to form a n′

-detection antibody;

n-capture antibody;

cTnT measurable assembly (D₁)(D₂) . . . (Dn′

) (C₁)(C₂) . . . (Cn)(cTnT), wherein the detection antibodies D are labeled with a detectable label comprising at least one acridinium compound, n′ and

n are independently an integer from 1 to 10, and antibodies C and D bind to (n+n′

) different epitopes of cTnT;

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) quantifying the amount of troponin-T in the test sample based on the measurement in step (e).

13. The immunoassay of claim 12, wherein the capture antibodies, the detection antibodies or capture and detection antibodies comprise humanized antibodies.

14. The immunoassay of claim 12, wherein the detection antibodies comprise humanized antibodies complexed with an anti-human IgG antibody.

15. The immunoassay of claim 14, wherein said anti-human IgG antibody is conjugated to the detectable label comprising at least one acridinium compound.

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

17. The immunoassay of claim 12, wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I:

18. The immunoassay of claim 12, wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

19. The immunoassay of claim 12, wherein the capture antibodies are immobilized on a solid phase.

20. The immunoassay of claim 19, 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.

21. The immunoassay of claim 12, wherein the capture antibodies are selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody.

22. The immunoassay of claim 12, wherein the detection antibodies are selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, and an affinity maturated antibody.

23. The immunoassay of claim 12, wherein the hydrogen peroxide is provided by adding a buffer or a solution containing hydrogen peroxide.

24. The immunoassay of claim 12, wherein the hydrogen peroxide is generated by adding a hydrogen peroxide generating enzyme to the test sample.

25. The immunoassay of claim 24, 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.

26. The immunoassay of claim 12, wherein the basic solution is a solution having a pH of at least about 10.

27. The immunoassay of claim 12, further comprising the step of quantifying the amount of the troponin-T in the test sample by relating the amount of light signal in step (e) to the amount of troponin-T in the test sample either by use of a standard curve for cTnT or by comparison to a reference standard.

28. The immunoassay of claim 27, comprising comparison to a reference standard comprising an anti-idiotypic antibody.

29. The immunoassay of claim 27, comprising comparison to a reference standard comprising a derivatized troponin-T.

30. The immunoassay of claim 29, wherein the derivatized troponin-T comprises troponin-T derivatized with a polyethylene glycol.

31. The immunoassay of claim 12, wherein the immunoassay is adapted for use in an automated system or semi-automated system.

32. A kit for quantifying the amount of troponin-T in a test sample, the kit comprising:

n capture antibodies (C) that bind to at least n epitopes on cTnT to form a n-capture antibody;

cTnT complex (C₁)(C₂) . . . (Cn)(cTnT), wherein n is an integer from 1 to 10;

n′

detection antibodies (D) that bind to n′

epitopes on cTnT to form a n′

-detection antibody;

n-capture antibody;

cTnT measurable assembly (D₁)(D₂) . . . (Dn′

) (C₁)(C₂) . . . (Cn)(cTnT), wherein n′ and

n are independently an integer from 1 to 10; and

wherein antibodies C and D bind to (n+n′

) different epitopes of cTnT; and

instructions for quantifying the amount of cTnT in the test sample.

33. The kit of claim 32, wherein the capture antibodies are immobilized on a solid phase.

34. The kit of claim 33, 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.

35. The kit of claim 32, further comprising a detectable label conjugated to the detection antibodies.

36. The kit of claim 35, wherein the detectable label is an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin.

37. The kit of claim 35, wherein the detectable label is an acridinium compound.

38. The kit of claim 37, wherein the acridinium compound is an acridinium-9-carboxamide having a structure according to formula I:

39. The kit of claim 37, wherein the acridinium compound is an acridinium-9-carboxylate aryl ester having a structure according to formula II:

40. The kit of claim 37, further comprising a basic solution.

41. The kit of claim 40, wherein the basic solution is a solution having a pH of at least about 10.

42. The kit of claim 37, further comprising a hydrogen peroxide source.

43. The kit of claim 42, wherein the hydrogen peroxide source comprises a buffer or a solution containing hydrogen peroxide.

44. The kit of claim 42, wherein the hydrogen peroxide source comprises a hydrogen peroxide generating enzyme.

45. The kit of claim 44, 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.

46. The kit of claim 32, wherein the capture antibodies, the detection antibodies or the capture and detection antibodies comprise humanized antibodies.

47. The kit of claim 32, wherein the detection antibodies comprise humanized antibodies complexed with an anti-human IgG antibody.

48. The kit of claim 47, wherein said anti-human IgG antibody is conjugated to a detectable label, wherein the detectable label is an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin.

49. The kit of claim 32, further comprising a cTnT reference standard.

50. The kit of claim 49, wherein the cTnT reference standard comprises an anti-idiotypic antibody.

51. The kit of claim 49, wherein the cTnT reference standard comprises a derivatized cTnT.

52. The kit of claim 51, wherein the derivatized cTnT comprises cTnT derivatized with a polyethylene glycol.