REAGENTS FOR REDUCING LEUKOCYTE INTERFERENCE IN IMMUNOASSAYS

• US 20110269159A1
• Filed: 04/30/2010
• Published: 11/03/2011
• Est. Priority Date: 04/30/2010
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Omakor, John Emegbero, Guhados, Ganesh, Campbell, John Lewis Emerson, Davis, Graham, Moss, Adam Roger
• Granted Patent: US 8,476,079 B2
• Time in Patent Office: Days
• US Class Current: 435/7.93
• CPC Class Codes: G01N 2333/475 Assays involving growth factors
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/5375 by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

Methods and devices for reducing interference from leukocytes in an analyte immunoassay are provided. In one embodiment, a method is provided comprising the steps of amending a biological sample such as a whole blood sample with one or more leukocidal reagents that reduce or eliminate the metabolic activity of leukocytes, and performing an immunoassay on the amended sample to determine the concentration of analyte in the sample. Preferably, the sample is amended with one or more enzymes and optionally one or more enzyme substrates and cofactors.

Claims

1. A method of performing an immunoassay for a target analyte in a blood sample comprising:

contacting a blood sample with a leukocidal reagent, wherein said reagent substantially inhibits activity of leukocytes in the sample; and

performing an immunoassay on the sample to detect a target analyte.

2. The method of claim 1, wherein the leukocidal reagent is a metabolic leukocidal reagent.

3. The method of claim 1, wherein the leukocidal reagent is non-ionic.

4. The method of claim 1, wherein the activity is phagocytosis.

5. The method of claim 1, wherein the reagent is an oxygen and glucose depleting reagent.

6. The method of claim 1, wherein the reagent is glucose oxidase.

7. The method of claim 1, wherein the reagent is glucose oxidase and glucose.

8. The method of claim 1, wherein the reagent is glucose oxidase and glucose, and said reagent mixes with the sample to provide a glucose oxidase activity above about 3 IU per mL and a glucose concentration above about 500 mg/dL.

9. The method of claim 1, wherein the reagent is glucose oxidase and an electron acceptor.

10. The method of claim 1, wherein the reagent comprises hexokinase and a source of adenosine-5′

-triphosphate (ATP).

11. The method of claim 1, wherein the reagent comprises hexokinase, creatine kinase, adenosine diphosphate (ADP), and creatine phosphate.

12. The method of claim 1, wherein the reagent comprises glucose dehydrogenase.

13. The method of claim 1, wherein the reagent comprises glucose dehydrogenase, NAD, NADH oxidase, and an electron acceptor.

14. The method of claim 1, wherein the reagent is an oxygen depleting reagent.

15. The method of claim 1, wherein the reagent is an oxygen depleting reagent selected from the group consisting of dithionite, glucose oxidase, and ascorbate oxidase.

16. The method of claim 1, wherein the reagent is a mitochondrial electron transport inhibitor.

17. The method of claim 1, wherein the reagent is a mitochondrial membrane uncoupling agent.

18. The method of claim 1, wherein the reagent is selected from the group consisting of cyanide, antimycin, rotenone, malonate, carbonyl cyanide p-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol, and oligomycin.

19. The method of claim 1, wherein the reagent is an amphipathic reagent.

20. The method of claim 1, wherein the reagent is an amphipathic reagent and is a saponin.

21. The method of claim 1, wherein the reagent is a leukocidin.

22. The method of claim 1, wherein the reagent is a mucopolysaccharide.

23. The method of claim 1, wherein the reagent is an AHN-1 antibody.

24. The method of claim 1, wherein the reagent is lipocortin-1.

25. The method of claim 1, wherein the immunoassay is a sandwich assay performed by contacting the sample with an immuno sensor comprising an immobilized first antibody to the target analyte, and a labeled second antibody to the target analyte.

26. The method of claim 1, wherein the immunoassay is a competitive assay performed by contacting the sample with an immuno sensor comprising an immobilized first antibody to the target analyte, and a labeled target analyte.

27. The method of claim 1, wherein the sample is contacted with beads opsonized for leukocytes.

28. The method of claim 1, wherein the reagent is glucose oxidase and glucose, and the sample is contacted with beads opsonized for leukocytes.

29. The method of claim 1, wherein the sample is a whole blood sample.

30. The method of claim 1, wherein the sample is amended with an anticoagulant.

31. The method of claim 1, wherein the analyte is selected from the group consisting of TnI, TnT, BNP, NTproBNP, proBNP, HCG, TSH, NGAL, theophylline, digoxin, and phenyloin.

32. A kit for performing an immunoassay for a target analyte in a whole blood sample comprising:

a leukocidal reagent wherein said reagent substantially inhibits activity of leukocytes in a sample, and

immunoassay reagents for detecting a target analyte.

33. The kit of claim 32, wherein the leukocidal reagent is a metabolic leukocidal reagent.

34. The kit of claim 32, wherein the leukocidal reagent is non-ionic.

35. The kit of claim 32, wherein the activity is phagocytosis.

36. The kit of claim 32, wherein the reagent is an oxygen and glucose depleting reagent.

37. The kit of claim 32, wherein the reagent is glucose oxidase.

38. The kit of claim 32, wherein the reagent is glucose oxidase and glucose.

39. The kit of claim 32, wherein the reagent is glucose oxidase and glucose, and said reagent mixes with the sample to give a glucose oxidase activity above about 3 IU per mL and a glucose concentration above about 500 mg/dL.

40. The kit of claim 32, wherein the reagent is glucose oxidase and an electron acceptor.

41. The kit of claim 32, wherein the reagent comprises hexokinase and a source of adenosine-5′

-triphosphate (ATP).

42. The kit of claim 32, wherein the reagent comprises hexokinase, creatine kinase, adenosine diphosphate (ADP), and creatine phosphate.

43. The kit of claim 32, wherein the reagent comprises glucose dehydrogenase.

44. The kit of claim 32, wherein the reagent comprises glucose dehydrogenase, NAD, NADH oxidase, and an electron acceptor.

45. The kit of claim 32, wherein the reagent is an oxygen depleting reagent.

46. The kit of claim 32, wherein the reagent is an oxygen depleting reagent selected from the group consisting of dithionite, glucose oxidase, and ascorbate oxidase.

47. The kit of claim 32, wherein the reagent is a mitochondrial electron transport inhibitor.

48. The kit of claim 32, wherein the reagent is a mitochondrial membrane uncoupling agent.

49. The kit of claim 32, wherein the reagent is selected from the group consisting of cyanide, antimycin, rotenone, malonate, carbonyl cyanide p-[trifluoromethoxyl]-phenyl-hydrozone, 2,4-dinitrophenol, and oligomycin.

50. The kit of claim 32, wherein the reagent is an amphipathic reagent.

51. The kit of claim 32, wherein the reagent is an amphipathic reagent and is a saponin.

52. The kit of claim 32, wherein the reagent is a leukocidin.

53. The kit of claim 32, wherein the reagent is a mucopolysaccharide.

54. The kit of claim 32, wherein the reagent is an AHN-1 antibody.

55. The kit of claim 32, wherein the reagent is lipocortin-1.

56. The kit of claim 32, wherein the immunoassay is a sandwich assay and wherein the immunoassay reagents comprise an immunosensor comprising an immobilized first antibody to the target analyte, and a labeled second antibody to the target analyte.

57. The kit of claim 32, wherein the immunoassay is a competitive assay and wherein the immunoassay reagents comprise an immunosensor comprising an immobilized first antibody to the target analyte, and a labeled target analyte.

58. The kit of claim 32, further comprising beads opsonized for leukocytes.

59. The kit of claim 32, wherein the reagent is glucose oxidase, and the sample is contacted with beads opsonized for leukocytes.

60. The kit of claim 32, wherein the sample is a whole blood sample.

61. The kit of claim 32, wherein the sample is amended with an anticoagulant.

62. The kit of claim 32, wherein the analyte is selected from the group consisting of TnI, TnT, BNP, NTproBNP, proBNP, HCG, TSH, NGAL, digoxin, theophylline and phenyloin.