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

• US 9,671,411 B2
• Filed: 04/06/2015
• Issued: 06/06/2017
• 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., Secretary of State for Health
• Inventors: Patel, Mehul, Poole, Stephen
• Primary Examiner(s): STANFIELD, CHERIE MICHELLE
• Application Number:
  US14/679,651
• Publication Number:
  US 20150285819A1
• Time in Patent Office: 792 Days
• 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:

(a) combining a monocyte-containing reagent and the sample to be tested in an 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;

(b) 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;

(c) adding an antibody to the cytokine or endogenous mediator of the inflammatory response to the assay system; and

(d) assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody;

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, comprising adding one or more beads or dipsticks coated with the antibody to the assay system.

3. The method of claim 1, comprising adding a detection antibody to the assay system.

4. 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

).

5. The method of claim 1, wherein the monocyte-containing reagent is PBMCs, cell line cells, or whole blood.

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

(a) 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;

(b) 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;

(c) transferring the contents of the first assay system to a second assay system which comprises an antibody to the cytokine or endogenous mediator of the inflammatory response; and

(d) assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody;

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

7. The method of claim 6, comprising adding a bead or dipstick coated with the antibody to the assay system.

8. The method of claim 6, comprising adding a detection antibody to the assay system.

9. The method of claim 6, 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

).

10. The method of claim 6, wherein the monocyte-containing reagent is PBMCs, cell line cells, or whole blood.

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

(a) combining a monocyte-containing reagent and the sample to be tested in an 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;

(b) incubating the monocyte-containing reagent and the sample,

(c) adding an antibody to a cytokine or endogenous mediator of the inflammatory response to the assay system; and

(d) assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody;

whereby the sample is tested.

12. The method of claim 11, comprising adding a bead or dipstick coated with the antibody to the assay system.

13. The method of claim 11, comprising adding a detection antibody to the assay system.

14. The method of claim 11, 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

).

15. The method of claim 11, wherein the monocyte-containing reagent is PBMCs, cell line cells, or whole blood.

16. A method of testing a sample comprising the steps of:

(a) 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;

(b) incubating the monocyte-containing reagent and the sample,

(c) transferring the contents of the first assay system to a second assay system which comprises an antibody to the cytokine or endogenous mediator of the inflammatory response; and

(d) assaying the assay system for the presence of cytokine or endogenous mediator bound to the antibody;

whereby the sample is tested.

17. The method of claim 16, comprising adding one or more beads or dipsticks coated with the antibody to the assay system.

18. The method of claim 16, comprising adding a detection antibody to the assay system.

19. The method of claim 18, wherein the detection antibody is an IL-6 antibody added to the second assay system.

20. The method of claim 16, 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

).

21. The method of claim 16, wherein the monocyte-containing reagent is PBMCs, cell line cells, or whole blood.

22. The method of claim 16, wherein (i) the monocyte-containing reagent is PBMCs, (ii) the cytokine is IL-6, (iii) the antibody is an anti-IL-6 antibody, (iv) the assay system is a microtiter plate, (v) wherein the method comprises an ELISA, or (vi) a combination thereof.

23. The method of claim 16, wherein the detection antibody is an anti-IL-6 antibody.

24. The method of claim 16, wherein (i) the second assay system is a microtiter plate, (ii) the antibody is an anti-IL-6 antibody, or (iii) a combination thereof.