Monocyte activation test better able to detect non-endotoxin pyrogenic contaminants in medical products

• US 7,736,863 B2
• Filed: 12/20/2006
• Issued: 06/15/2010
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: Baxter Healthcare SA (Baxter International Inc.), Baxter International Inc., Health Protection Agency (Government of United Kingdom), Secretary of State for Health
• Original Assignee: National Institute for Biological Standards and Control (NIBSC)
• Inventors: Patel, Mehul, Poole, Stephen
• Primary Examiner(s): STANFIELD, CHERIE MICHELLE
• Application Number:
  US11/613,866
• Publication Number:
  US 20070184496A1
• Time in Patent Office: 1,273 Days
• Field of Search: None
• US Class Current: 435/7.21
• CPC Class Codes: G01N 2333/5412 IL-6
• CPC Class Codes: G01N 2800/7095 Inflammation
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/5055 involving macrophages
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• CPC Class Codes: G01N 33/6869 Interleukin

Abstract

An improved monocyte activation test is described that is better able to detect non-endotoxin pyrogens in medical products, in which a sample is incubated with a monocyte-containing reagent in an assay system comprising at least one surface comprising polypropylene. The invention also concerns assay systems for use in these tests that include at least one microtiter well having at least one interior surface comprising polypropylene and having a shape such that monocyte-containing reagent is concentrated in the well to provide greater cell to cell contact. The invention also relates to a diagnostic kit that can be used to test for the presence of non-endotoxin pyrogens in a sample.

Claims

1. A method of detecting non-endotoxin pyrogens in a sample comprising the steps of:

combining a monocyte-containing reagent and the sample to be tested in a first assay system which includes at least one microtiter well shaped such that the monocyte-containing reagent is concentrated to provide greater cell to cell contact as compared to a flat-bottomed microtiter well, wherein the surface of the microtiter well comprises a polypropylene coating or the entire microtiter well is composed of polypropylene;

incubating the monocyte-containing reagent and the sample, wherein, when the sample comprises a pyrogen, the monocyte-containing reagent produces a cytokine or an endogenous mediator of the inflammatory response;

transferring the contents of the first assay system to a second assay system which comprises at least one surface treated with an antibody to the cytokine or endogenous mediator of the inflammatory response; and

assaying the second assay system for the presence of cytokine or endogenous mediator bound to the antibody on the surface;

whereby an elevated level of cytokine or endogenous mediator bound to the surface indicates the presence of the pyrogens in the sample tested.

2. The method of claim 1 wherein the cytokine is selected from the group consisting of interleukin-1 (IL-1), interleukin-1ra (IL-1ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α

(TNF-α

).

3. The method of claim 2 wherein the cytokine is IL-6.

4. The method of claim 1 wherein the endogenous mediator is endothelin.

5. The method of claim 1 wherein the sample is a medical product for parenteral administration.

6. The method of claim 5 wherein the medical product is a blood product, dialysis solution, vaccine, or plasma expander.

7. The method of claim 1 wherein the microtiter well comprises an open top, an upper region extending downwardly from the open top, and a bottom wall tapering in diameter from a location above the lowest point to the lowest point.

8. The method of claim 1 wherein the microtiter well comprises an open top, an upper region extending downwardly from the open top having a top end and a bottom end, and a bottom region having a top end and a lowest point, the bottom region extending from the bottom end of the upper region and tapering in diameter more rapidly than the upper region from the top end of the bottom region toward the lowest point.

9. The method of claim 7 wherein the bottom wall of said microtiter well is non-planar, curved, parabolic or downwardly extending, or the sides of said microtiter well are sloped inward.

10. The method of claim 1 wherein the assay system(s) is a colorimetric enzyme-linked immunosorbent assay (ELISA), a radio-labeled immunoassay, or a fluorescence-labeled immunoassay.

11. The method of claim 1, wherein the monocyte-containing reagent comprises peripheral blood mononuclear cells (PBMCs) or monocytic cell line cells.

12. The method of claim 11, wherein the monocytic cell line comprises a human monocytic cell line.

13. The method of claim 12, wherein the monocytic cell line is MONOMAC-6, THP-1, or 28SC.

14. The method of claim 11, wherein the monocytic cell line comprises a cell line having endogenous CD14 and Toll-like receptors or which has been transfected with CD14 and/or Toll-like receptors and/or reporter genes for inflammatory or pyrogenic mediators.

15. The method of claim 11, wherein the PBMCs are human PBMCs.

16. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density which is higher than the cell density of monocytes in whole blood.

17. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 250,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

18. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 500,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

19. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 600,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

20. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 700,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

21. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 800,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

22. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 900,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

23. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,000,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

24. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,100,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

25. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,200,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

26. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,300,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

27. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,400,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

28. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,500,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

29. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,600,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

30. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,700,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

31. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,800,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

32. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,900,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

33. The method of claim 11, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 2,000,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

34. The method of claim 1, wherein the monocyte-containing reagent comprises whole blood.

35. The method of claim 34, wherein the whole blood comprises human whole blood.

36. A method of detecting non-endotoxin pyrogens in a sample comprising the steps of:

combining a monocyte-containing reagent and the sample to be tested in an assay system comprising (i) at least one microtiter well shaped such that the monocyte-containing reagent is concentrated to provide greater cell to cell contact as compared to a flat-bottomed microtiter well, wherein the surface of the microtiter well comprises a polypropylene coating or the entire microtiter well is composed of polypropylene, and (ii) at least one surface treated with an antibody to a cytokine or an endogenous mediator of the inflammatory response;

incubating the monocyte-containing reagent and the sample, wherein, when the sample comprises a pyrogen, the monocyte-containing reagent produces a cytokine or an endogenous mediator of the inflammatory response, and

assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody on the surface;

whereby an elevated level of cytokine or endogenous mediator bound to the surface indicates the presence of the pyrogens in the sample tested.

37. The method of claim 36, wherein the cytokine is selected from the group consisting of interleukin-1 (IL-1), interleukin-1ra (IL-1ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α

(TNF-α

).

38. The method of claim 37 wherein the cytokine is IL-6.

39. The method of claim 36 wherein the endogenous mediator is endothelin.

40. The method of claim 36 wherein the sample is a medical product for parenteral administration.

41. The method of claim 40 wherein the medical product is a blood product, dialysis solution, vaccine, or plasma expander.

42. The method of claim 36, wherein the microtiter well comprises an open top, an upper region extending downwardly from the open top, and a bottom wall tapering in diameter from a location above the lowest point to the lowest point.

43. The method of claim 36 wherein the microtiter well comprises an open top, an upper region extending downwardly from the open top having a top end and a bottom end, and a bottom region having a top end and a lowest point, the bottom region extending from the bottom end of the upper region and tapering in diameter more rapidly than the upper region from the top end of the bottom region toward the lowest point.

44. The method of claim 42 wherein the bottom wall of said microtiter well is non-planar, curved, parabolic or downwardly extending, or the sides of said microtiter well are sloped inward.

45. The method of claim 36 wherein the assay system(s) is a colorimetric enzyme-linked immunosorbent assay (ELISA), a radio-labeled immunoassay, or a fluorescence-labeled immunoassay.

46. The method of claim 36, wherein the monocyte-containing reagent comprises peripheral blood mononuclear cells (PBMCs) or monocytic cell line cells.

47. The method of claim 46, wherein the monocytic cell line comprises a human monocytic cell line.

48. The method of claim 47, wherein the monocytic cell line is MONOMAC-6, THP-1, or 28SC.

49. The method of claim 46, wherein the PBMCs are human PBMCs.

50. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density which is higher than the cell density of monocytes in whole blood.

51. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 250,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

52. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 500,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

53. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 600,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

54. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 700,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

55. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 800,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

56. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 900,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

57. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,000,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

58. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,100,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

59. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,200,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

60. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,300,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

61. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,400,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

62. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,500,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

63. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,600,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

64. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,700,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

65. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,800,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

66. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 1,900,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

67. The method of claim 46, wherein the monocyte-containing reagent is present in said assay system at a cell density of at least or about 2,000,000 cells per well, wherein the cell density does not exceed a maximum cell density at which insufficient cell nutrients exist in the volume of reagent within the well to properly maintain the cells in the well.

68. The method of claim 36, wherein the monocytic cell line comprises a cell line having endogenous CD14 and Toll-like receptors or which has been transfected with CD14 and/or Toll-like receptors and/or reporter genes for inflammatory or pyrogenic mediators.

69. The method of claim 36, wherein the monocyte-containing reagent comprises whole blood.

70. The method of claim 69, wherein the whole blood comprises human whole blood.