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

• US 8,053,200 B2
• Filed: 04/26/2010
• Issued: 11/08/2011
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: Baxter Healthcare SA (Baxter International Inc.), Baxter International Inc., Secretary of State for Health
• Original Assignee: Baxter Healthcare SA (Baxter International Inc.), Baxter International Inc., National Institute for Biological Standards and Control (NIBSC)
• Inventors: Patel, Mehul, Poole, Stephen
• Primary Examiner(s): Woodward; Cherie M
• Application Number:
  US12/767,537
• Publication Number:
  US 20100203551A1
• Time in Patent Office: 561 Days
• Field of Search: None
• US Class Current: 435/7.1
• 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 testing a sample, comprising the steps of:

combining a monocyte-containing reagent and a 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;

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 the sample is tested.

2. The method of claim 1, wherein the sample is a pharmaceutical, nutrient, or medical product.

3. The method of claim 1, wherein the cytokine or endogenous mediator of the inflammatory response is a proinflammatory cytokine or a Th1 cytokine.

4. The method of claim 3, wherein cytokine or endogenous mediator of the inflammatory response is TNF-alpha, IL-2, IFN, or IL-8.

5. 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.

6. 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.

7. The method of claim 5, 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.

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

9. The method of claim 8, wherein the monocytic cell line comprises a human monocytic cell line selected from the group consisting of:

MONOMAC-6, THP-1, 28SC, and 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.

10. The method of claim 1, wherein the monocyte-containing reagent comprises a number of cells sufficient to provide at least or about 125,000 cells per well of the first assay system.

11. A method of testing a sample, comprising the steps of:

combining a monocyte-containing reagent and a sample to be tested in a first 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 the cytokine or endogenous mediator of the inflammatory response;

incubating the monocyte-containing reagent and the sample; and

assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody on the surface, whereby the sample is tested.

12. The method of claim 11, wherein the sample is a pharmaceutical, nutrient, or medical product.

13. The method of claim 11, wherein the cytokine or endogenous mediator of the inflammatory response is a proinflammatory cytokine or a Th1 cytokine.

14. The method of claim 13, wherein cytokine or endogenous mediator of the inflammatory response is TNF-alpha, IL-2, IFN, or IL-8.

15. The method of claim 11, 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.

16. The method of claim 11, 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.

17. The method of claim 15, 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.

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

19. The method of claim 18, wherein the monocytic cell line comprises a human monocytic cell line selected from the group consisting of:

MONOMAC-6, THP-1, 28SC, and 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.

20. The method of claim 11, wherein the monocyte-containing reagent comprises a number of cells sufficient to provide at least or about 125,000 cells per well of the assay system.