Methods for quantifying IL-33

Patent metadata

Field	Value
Publication number	US-12092634-B2
Application number	US-201917046200-A
Country	US
Kind code	B2
Filing date	Apr 10, 2019
Priority date	Apr 11, 2018
Publication date	Sep 17, 2024
Grant date	Sep 17, 2024

Abstract

Methods and compositions for detecting and quantifying cytokines are provided. The disclosed assays have reduced assay interference relative to commercially available assays and/or a control assay. The interference can be cytokine dependent, cytokine independent, or both. One embodiment provides an IL-33 immunoassay that reduces assay interference caused by endogenous soluble IL-33 binding molecules present in the sample. Exemplary soluble IL-33 binding molecules include, but are not limited to anti-IL-33 antibodies, soluble ST2 receptor, and serum components. In some embodiments a blocking agent is added to the sample to reduce, inhibit, or block IL-33 complexes in the sample from reforming after acid dissociation of the IL-33 complexes in the sample. In one embodiment, the blocking agent and the detection reagent do not compete for binding to IL-33.

First claim

We claim:

1. A method of decreasing IL-33 assay interference, comprising the steps of:
acidifying a sample comprising blood, serum, or plasma to a pH of 3 to 5 to dissociate IL-33 from IL-33 complexes in the sample, wherein acidifying the sample includes adding a denaturing agent to the sample;
subsequently neutralizing the acidified sample with a buffered basic solution comprising a detection reagent, wherein the detection reagent does not compete for binding to IL-33;
adding a capture reagent to the sample; and
detecting the detection reagent, wherein the quantity of detection reagent detected correlates to the quantity of IL-33 in the sample.

2. The method of claim 1 , wherein the sample is from a subject that was administered an IL-33 drug product.

3. The method of claim 2 , wherein the IL-33 drug product comprises an anti- IL-33 antibody.

4. The method of claim 1 , wherein the IL-33 complex comprises IL-33 non-covalently bound to a protein.

5. The method of claim 4 , wherein the protein is an endogenous serum protein.

6. The method of claim 4 , wherein the protein is ST2 or an IL-33 binding fragment thereof.

7. The method of claim 4 , where the protein is an anti-IL-33 antibody or an IL-33 binding fragment thereof.

8. The method of claim 1 , wherein the sample is acidified for 5 to 60 minutes.

9. The method of claim 1 , wherein the detection reagent comprises an anti-IL-33 antibody conjugated to a detectable label.

10. The method of claim 1 , wherein the detection reagent comprises a detectable agent selected from the group consisting of a rare transition metal particle, a fluorophore, a chromophore, a quantum dot, and noble metal nanoparticles.

11. The method of claim 1 , wherein the detection reagent is labeled with ruthenium.

12. The method of claim 1 , wherein the capture reagent is conjugated to solid support.

13. The method of claim 1 , wherein the capture reagent is biotinylated.

14. The method of claim 1 , wherein the sample is acidified with acetic acid.

15. The method of claim 1 , wherein the buffered basic solution further comprises a blocking agent that inhibits IL-33 complex formation, and wherein the blocking agent does not compete for binding to IL-33.

16. The method of claim 15 , wherein the blocking agent is an anti-ST2 antibody.

17. The method of claim 1 , wherein the capture reagent is added to the sample during the acidification step.

18. The method of claim 1 , wherein the blocking agent is added to the sample after the neutralizing step.

19. The method of claim 1 , wherein the capture reagent and the blocking agent are added to the sample after the neutralizing step.

20. The method of claim 1 , wherein the sample is obtained from a subject diagnosed with or suspected of having an inflammatory disease or disorder.

21. The method of claim 1 , wherein the sample is obtained from a subject diagnosed with or suspected of having asthma, chronic obstructive pulmonary disease, or atopic dermatitis.

22. The method of claim 1 , wherein the solid support is an electrochemiluminescence platform.

23. The method of claim 1 , wherein the quantity of IL-33 is determined by correlating the amount of detected detection reagent to a predetermined reference standard.

24. The method of claim 1 , wherein the denaturing agent is urea.

25. A method of quantifying interleukin- 33 in a serum sample, comprising the steps of:
acidifying the serum sample to a pH of 3 to 5 to dissociate IL-33 complexes in the sample, wherein acidifying the serum sample includes adding a denaturing agent to the serum sample;
neutralizing the acidified sample with a buffered basic solution comprising an anti-human IL-33 antibody labeled with a detectable label;
adding the sample to an avidin-coated solid support comprising a biotinylated anti-human IL-33 antibody; and
detecting the detectable label on the avidin-coated solid support, wherein the quantity of detectable label detected correlates to the quantity of IL-33 in the sample.

26. The method of claim 25 , wherein the solid support is a streptavidin-coated electrochemiluminescence platform.

27. The method of claim 25 , wherein the detectable label is ruthenium.

28. The method of claim 25 , wherein the buffered basic solution further comprises an anti-human ST2 antibody.

29. The method of claim 25 , wherein the denaturing agent is urea.

30. The method of claim 28 , wherein the anti-human ST2 antibody is present in an amount sufficient to reduce ST2 binding to IL-33 in the sample .

Assignees

• Regeneron Pharma

Inventors

• Partridge Michael
• Oliveira Sumner Giane
• Zylstra Joshua

Classifications

• G01N33/6869 Primary

  Interleukin · CPC title

• G01N33/5306 Primary

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N2333/54

  Interleukins [IL] · CPC title

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title