MONOCYTE ACTIVATION TEST BETTER ABLE TO DETECT NON-ENDOTOXIN PYROGENIC CONTAMINANTS IN MEDICAL PRODUCTS

• US 20070184496A1
• Filed: 12/20/2006
• Published: 08/09/2007
• 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: Baxter Healthcare SA (Baxter International Inc.), Baxter International Inc., National Biological Standards Board
• Inventors: Patel, Mehul, Poole, Stephen
• Granted Patent: US 7,736,863 B2
• Time in Patent Office: Days
• US Class Current: 435/7.210
• 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 comprises at least one surface comprising polypropylene, wherein the monocyte-containing reagent comprises peripheral blood mononuclear cells or monocytic cell line cells, is in contact with said surface, and is present in said assay system at a high cell density;

incubating the monocyte-containing reagent and the sample, wherein 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 a cytokine or an 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 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 at least one of the first and second assay system includes at least one microtiter well, and the surface(s) is at least a portion of the interior of the microtiter well.

8. The method of claim 7 wherein the well is shaped such that the monocyte-containing reagent is concentrated to provide greater cell to cell contact as compared to a flat-bottomed microtiter well.

9. The method of claim 7 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.

10. The method of claim 7 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.

11. The method of claim 9 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.

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

13. The method of claim 1 wherein the monocytic cell line comprises a human monocytic cell line.

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

15. The method of claim 1 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.

16. The method of claim 1 wherein the monocyte-containing reagent is present in said assay system at a cell density of at least about 250,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000 or 2,000,000 cells per well.

17. The method of claim 1 wherein the peripheral blood mononuclear cells comprise human peripheral blood mononuclear cells.

18. A method of detecting non-endotoxin pyrogens in a parenterally administered medical product comprising the steps of:

combining a monocyte-containing reagent and the medical product to be tested in a first assay system which comprises at least one surface comprising polypropylene, wherein the monocyte-containing reagent comprises whole blood and is in contact with said surface;

incubating the monocyte-containing reagent and the medical product, wherein the monocyte-containing reagent produces IL-6;

transferring the contents of the first assay system to a second assay system which comprises at least one surface treated with an antibody to IL-6; and

assaying the second assay system for the presence of IL-6 bound to the antibody on the surface;

whereby an elevated level of IL-6 bound to the surface indicates the presence of pyrogens in the medical product tested.

19. The method of claim 18 wherein the monocyte-containing reagent is present in said assay system at a cell density of greater than about 100,000, 200,000, 210,000, 220,000, 230,000, 240,000, 250,000, 260,000, 270,000, 280,000, 290,000, 300,000, 310,000, 320,000, 330,000, 340,000, 350,000, 360,000, 370,000, 380,000, 390,000 or 400,000 peripheral blood mononuclear cells per well.

20. The method of claim 18 wherein the monocyte-containing reagent further comprises a diluent.

21. The method of claim 18 wherein the monocyte-containing reagent further comprises an anticoagulant.

22. The method of claim 18 wherein the whole blood comprises human whole blood.

23. A method of detecting non-endotoxin pyrogens in a parenterally administered medical product comprising the steps of:

combining a monocyte-containing reagent and the medical product to be tested in an assay system which comprises at least one surface comprising polypropylene and at least one surface treated with an antibody to IL-6, wherein the monocyte-containing reagent comprises whole blood and is in contact with said surface comprising polypropylene; and

assaying the assay system for the presence of IL-6 bound to the antibody on the surface;

whereby an elevated level of IL-6 bound to the surface indicates the presence of pyrogens in the medical product tested.

24. The method of claim 23 wherein the assay system includes at least one microtiter well, and the surface(s) is at least a portion of the interior of the microtiter well.

25. A method of culturing cells contained within a monocyte-containing reagent for use in a non-endotoxin pyrogen assay comprising the step of:

combining the monocyte-containing reagent and a sample to be tested in an assay system which comprises at least one surface comprising polypropylene, wherein the monocyte-containing reagent comprises peripheral blood mononuclear cells or monocytic cell line cells, is in contact with said surface, and is present in said assay system at a high cell density.

26. The method of claim 25 wherein the assay system comprises at least one microtiter well shaped such that the monocyte-containing reagent is concentrated as compared to a flat-bottomed microtiter well, at least one surface of said well comprises polypropylene, and the monocyte-containing reagent is present in such well at a high cell density.

27. The method of claim 25 further comprising the step of 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 pyrogens in the sample tested.

28. The method of claim 25 wherein the assay system comprises at least one surface treated with an antibody to a cytokine, and the surface on which the antibody is applied is at least a portion of the interior of said microtiter well.

29. A diagnostic kit comprising:

a microtiter plate comprising a plurality of microtiter wells shaped such that cells contained in each well are concentrated as compared to a flat-bottomed microtiter well, a surface of said well comprising polypropylene; and

a cryopreserved monocyte-containing reagent which is contained in the wells of said plate, wherein the monocyte-containing reagent comprises cryopreserved peripheral blood mononuclear cells or cryopreserved monocytic cell line cells, is in contact with the surfaces of said wells, and is present in said well at a high cell density.

30. The diagnostic kit of claim 29 wherein said well further comprises a barrier which is impervious to monocytes.

31. The diagnostic kit of claim 30 wherein said barrier comprises a membrane, a grid, a sieve, or a sifter.

32. A diagnostic kit comprising:

an assay system for detecting non-endotoxin pyrogens in a parenterally administered medical product, said assay system comprising a microtiter plate comprising a plurality of microtiter wells shaped such that cells contained in each well are concentrated as compared to a flat-bottomed microtiter well, a surface of said well comprising polypropylene;

a cryopreserved monocyte-containing reagent which is contained in the wells of said plate, wherein the monocyte-containing reagent comprises cryopreserved whole blood and is in contact with the surfaces of said wells; and

an antibody to IL-6.