Assays for the detection of anti-TNF drugs and autoantibodies
View Patent ↗The present invention provides assays for detecting and measuring the presence or level of anti-TNFα drug therapeutics and autoantibodies in a sample. The present invention is useful for optimizing therapy and monitoring patients receiving anti-TNFα drug therapeutics to detect the presence or level of autoantibodies (e.g., HACA and/or HAHA) against the drug.
1. A method for detecting the presence or level of an autoantibody to an anti-TNFα drug in a sample, the method comprising:
(a) contacting a labeled anti-TNFα drug and labeled TNFα with the sample to form a first labeled complex between the labeled anti-TNFα drug, the labeled TNFα, and the autoantibody and a second labeled complex between the labeled anti-TNFα drug and the autoantibody, wherein the labeled anti-TNFα drug and the labeled TNFα comprise different labels;
(b) subjecting the first labeled complex and the second labeled complex to size exclusion chromatography to separate the first labeled complex and the second labeled complex; and
(c) detecting the first labeled complex and the second labeled complex, thereby detecting the presence or level of a non-neutralizing form of the autoantibody when both the first labeled complex and the second labeled complex are present, and detecting the presence or level of a neutralizing form of the autoantibody when only the second labeled complex is present.
2. The method of claim 1 , wherein the sample is serum.
3. The method of claim 1 , wherein the anti-TNFα drug is a member selected from the group consisting of infliximab, etanercept, adalimumab, certolizumab pegol, and combinations thereof.
4. The method of claim 1 , wherein the autoantibody is a member selected from the group consisting of a human anti-mouse antibody (HAMA), a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA) and combinations thereof.
5. The method of claim 1 , wherein the size exclusion chromatography is size exclusion-high performance liquid chromatography (SE-HPLC).
6. The method of claim 1 , wherein the labeled anti-TNFα drug is a fluorophore-labeled anti-TNFα drug.
7. The method of claim 6 , wherein the fluorophore is selected from the group consisting of a polymethine dye, a pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene dye, a rhodamine dye, a coumarin dye, and a fluorescein dye.
8. The method of claim 1 , wherein the labeled TNFα is a fluorophore-labeled TNFα.
9. The method of claim 8 , wherein the fluorophore is selected from the group consisting of a polymethine dye, a pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene dye, a rhodamine dye, a coumarin dye, and a fluorescein dye.
10. The method of claim 1 , wherein the sample is obtained from a subject receiving therapy with the anti-TNFα drug.
11. A method for detecting the presence or level of an autoantibody to an anti-TNFα drug in a sample, the method comprising:
(a) contacting a labeled anti-TNFα drug with the sample to firm a first labeled complex between the labeled anti-TNFα drug and the autoantibody;
(b) subjecting the first labeled complex to a first size exclusion chromatography to separate the first labeled complex from free labeled anti-TNFα drug;
(c) detecting the first labeled complex, thereby detecting the presence or level of the autoantibody;
(d) contacting labeled TNFα with the first labeled complex from step (c) to form a second labeled complex between the labeled anti-TNFα drug and the labeled TNFα, wherein the labeled anti-TNFα drug and the labeled TNFα comprise different labels, and wherein the second labeled complex does not include the autoantibody;
(e) subjecting the second labeled complex to a second size exclusion chromatography to separate the second labeled complex from free labeled TNFα; and
(f) detecting the second labeled complex, thereby detecting the presence or level of a neutralizing form of the autoantibody.
12. The method of claim 11 , wherein the sample is serum.
13. The method of claim 11 , wherein the anti-TNFα drug is a member selected from the group consisting of infliximab, etanercept, adalimunab, certolizumab pegol, and combinations thereof.
14. The method of claim 11 , wherein the autoantibody is a member selected from the group consisting of a human anti-mouse antibody (HAMA), a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), and combinations thereof.
15. The method of claim 11 , wherein the first and/or the second size exclusion chromatography is size exclusion-high performance liquid chromatography (SE-HPLC).
16. The method of claim 11 , wherein the labeled anti-TNFα drug is a fluorophore-labeled anti-TNFα drug.
17. The method of claim 16 , wherein the fluorophore is selected from the group consisting of a polymethine dye, a pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene dye, a rhodamine dye, a coumarin dye, and a fluorescein dye.
18. The method of claim 11 , wherein the labeled TNFα is a fluorophore-labeled TNFα.
19. The method of claim 18 , wherein the fluorophore is selected from the group consisting of a polymethine dye, a pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene dye, a rhodamine dye, a coumarin dye, and a fluorescein dye.
20. The method of claim 11 , wherein the sample is obtained from a subject receiving therapy with the anti-TNFα drug.