Pyrogenicity test for use with automated immunoassay systems

• US 20010034037A1
• Filed: 12/01/2000
• Published: 10/25/2001
• Est. Priority Date: 12/03/1999
• Status: Active Grant

• Current Assignee: Baxter International Inc., Health Protection Agency (Government of United Kingdom), Secretary of State for Health
• Original Assignee: National Institute For Biological Standards And Co.
• Inventors: Patel, Mehul, Poole, Stephen
• Granted Patent: US 6,696,261 B2
• Time in Patent Office: Days
• US Class Current: 435/7.21
• 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

The invention concerns an improved pyrogenicity test for use in automated immunoassay systems, in which a sample is incubated with a monocyte-containing reagent in a pyrogen-free assay system preferably comprising a surface coated with pyrogen-free anti-cytokine antibodies. The invention also concerns pyrogen-free assay systems coated with pyrogen-free antibodies for use in these tests. The invention also relates to a test to measure the basal production of endogenous mediators of the inflammatory response by leukocytes and a method of measuring the capacity of a leukocyte to respond to a stimuli.

Claims

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

a) combining a pyrogen-free monocyte-containing reagent and the sample to be tested in a pyrogen-free assay system, wherein the assay system comprises at least one surface treated with a pyrogen-free antibody to a cytokine; and

b) assaying the assay system for the presence of cytokine bound to the antibody on the surface;

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

2. The method of

claim 1

wherein the monocyte-containing reagent comprises whole blood.

3. The method of

claim 2

wherein the monocyte-containing reagent further comprises a diluent.

4. The method of

claim 2

wherein the monocyte-containing reagent further comprises an anticoagulant.

5. The method of

claim 4

wherein the monocyte-containing reagent further comprises a diluent.

6. The method of

claim 3

wherein the diluent is RPMI media.

7. The method of

claim 2

wherein the whole blood is human whole blood.

8. The method of

claim 1

wherein the cytokine is selected from the group consisting of interleukin-1, interleukin-1ra, interleukin-6, interleukin-8, tumor necrosis factor-α

, and prostaglandin E₂.

9. The method of

claim 8

wherein the cytokine is interleukin-6.

10. The method of

claim 1

wherein the assay system includes at least one microtiter well, and the surface on which the antibody is applied is a portion of the interior of the microtiter well.

11. The method of

claim 10

wherein the microtiter well is made from polystyrene or polypropylene.

12. The method of

claim 1

wherein the assay system is assayed by a colorimetric enzyme-linked immunosorbent assay for the cytokine.

13. The method of

claim 1

wherein the assay system is assayed by a radio-labeled immunoassay for the cytokine.

14. The method of

claim 1

wherein the assay system is assayed by a fluorescence-labeled immunoassay for the cytokine.

15. The method of

claim 1

wherein the pyrogen-free antibody is a polyclonal antibody.

16. The method of

claim 1

wherein the pyrogen-free antibody is a monoclonal antibody.

17. The method of

claim 1

wherein the sample is a medicinal drug for injection or infusion.

18. The method of

claim 1

wherein the sample is a blood product.

19. A pyrogen-free assay system wherein the assay system comprises a surface coated with a pyrogen-free antibody to a cytokine.

20. The pyrogen-free assay system of

claim 19

wherein the assay system further comprises at least one microtiter well, and wherein the surface to which the pyrogen-free antibody is applied is a portion of the interior of the microtiter well.

21. The pyrogen-free assay system of

claim 20

wherein the well is made from polystyrene or polypropylene.

22. The pyrogen-free assay system of

claim 20

wherein the well is part of a planar array of similar wells, so situated so that the array of wells may be read with automated reading equipment.

23. The pyrogen-free assay system of

claim 19

wherein the cytokine is selected from the group consisting of interleukin-1, interleukin-1ra, interleukin-6, interleukin-8, tumor necrosis factors-α

, and prostaglandin E₂.

24. The pyrogen-free assay system of

claim 19

wherein the cytokine is interleukin-6.

25. The pyrogen-free assay system of

claim 19

wherein the pyrogen-free antibody is a polyclonal antibody.

26. The pyrogen-free assay system of

claim 19

wherein the pyrogen-free antibody is a monoclonal antibody.

27. A method of detecting pyrogens in a sample comprising the steps of:

a) combining a pyrogen-free monocyte-containing reagent and the sample to be tested in a pyrogen-free assay system, wherein the assay system comprises at least one surface treated with a pyrogen-free antibody to an endogenous mediator of the inflammatory response; and

b) assaying the assay system for the presence of the mediator bound to the antibody on the surface;

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

28. The method of

claim 27

wherein the endogenous mediator of the inflammatory response is endothelin.

29. A method of measuring the capacity of a leukocyte to respond to a stimuli comprising the steps of:

a) combining a pyrogen-free leukocyte-containing reagent and a stimuli in a pyrogen-free assay system, wherein the assay system comprises at least one surface treated with a pyrogen-free antibody to an endogenous mediator of the inflammatory response; and

b) assaying the assay system for the presence of the mediator bound to the antibody on the surface;

whereby an elevated level of the mediator bound to the surface indicates the response of the leukocyte to the stimuli.

30. The method of

claim 29

wherein the stimuli is selected from the group consisting of endotoxin, an infectious agent, vaccine, adjuvant, and drug.

31. The method of

claim 29

wherein the leukocyte-containing reagent comprises whole human blood.

32. The method of

claim 29

wherein the endogenous mediator of the inflammatory response comprises a cytokine.

33. A method of measuring the basal production of endogenous mediators of the inflammatory response by leukocytes comprising the steps of:

a) adding a pyrogen-free leukocyte-containing reagent to a pyrogen-free assay system, wherein the assay system comprises at least one surface treated with a pyrogen-free antibody to an endogenous mediator of the inflammatory response; and

b) assaying the assay system for the presence of the mediator bound to the antibody on the surface;

whereby the amount of mediator bound to the surface indicates the basal production of endogenous mediators of the inflammatory response by the leukocytes.

34. The method of

claim 33

wherein the leukocyte-containing reagent comprises whole human blood.

35. The method of

claim 33

wherein the endogenous mediator of the inflammatory response comprises a cytokine.