CD40L-binding agent based platelet aggregation assays
View Patent ↗The present invention provides methods of determining platelet aggregation, methods of determining susceptibility to clotting upon administration of a CD40L-binding moiety, and kits related thereto.
1. An in vitro method of assaying platelet aggregation comprising:
providing a sample comprising platelets obtained from a human subject;
contacting the sample with a CD40L-binding moiety;
contacting the sample with a cross-linking agent capable of cross-linking the CD40L binding moiety;
contacting the sample with a platelet activating agent; and
determining the amount of platelet aggregation in the sample, wherein sedimented platelets is indicative of aggregation of the platelets.
2. The method of claim 1 , wherein the CD40L-binding moiety is an antibody or an antigen-binding fragment thereof.
3. The method of claim 1 , wherein the cross-linking agent is selected from the group consisting of sCD40L, anti-human IgG antibody, anti-hFc antibody, rheumatoid factor (RF), Fc receptor-positive accessory cell, soluble protein A, and soluble human Fc receptor.
4. The method of claim 1 , wherein the platelet activating agent is in an amount suitable to induce a sub-optimal amount of platelet aggregation in the sample.
5. The method of claim 4 , wherein the sub-optimal amount of platelet aggregation is less than 70% platelet aggregation.
6. The method of claim 4 , wherein a titration is first performed on the platelets to determine the amount of platelet activating agent suitable to induce a sub-optimal amount of platelet aggregation in the sample.
7. The method of claim 1 , wherein the platelet activating agent is selected from the group consisting of ADP, collagen, thrombin, thromboxane, neutrophil elastase, p-selectin and convulxin.
8. The method of claim 7 , wherein the platelet activating agent is ADP and wherein the ADP is present in the sample at a final concentration of 1 μM.
9. The method of claim 2 , wherein the ratio of anti-CD40L antibody or antigen-binding fragment thereof to cross-linking agent is 1:1000 to 1000:1, 1:500 to 500:1 or 3:2.
10. The method of claim 1 , wherein the sample comprising platelets is obtained from platelet rich plasma.
11. The method of claim 1 , wherein the CD40L-binding moiety and the cross-linking agent are mixed prior to addition to the sample comprising platelets.
12. The method of claim 11 , wherein the CD40L-binding moiety and the cross-linking agent are mixed for no less than 20 minutes prior to addition to the sample comprising platelets.
13. The method of claim 1 , wherein the CD40L-binding moiety and the cross-linking agent are not a preformed immune complex.
14. The method of claim 1 , wherein the amount of platelet aggregation in the sample is compared to the amount of platelet aggregation in a control sample.
15. The method of claim 14 , wherein the control sample is contacted with an anti-CD40L antibody or antigen-binding fragment thereof or with a control human IgG.
16. The method of claim 2 , wherein the anti-CD40L antibody or antigen-binding fragment thereof is hu5c8 or an antigen-binding fragment thereof.
17. An in vitro method of assaying platelet aggregation comprising:
providing a sample comprising platelets obtained from a human subject;
contacting the sample with an hu5c8 antibody or antigen binding fragment thereof;
contacting the sample with a cross-linking agent capable of cross-linking the hu5c8 antibody or antigen-binding fragment thereof; wherein the cross-linking agent is selected from the group consisting of sCD40L and anti-hFc antibody;
contacting the sample with a ADP;
determining the amount of platelet aggregation in the sample; wherein sedimented platelets is indicative of aggregation of the platelets.
18. The method of claim 17 , wherein the ADP is in an amount suitable to induce a sub-optimal amount of platelet aggregation in the sample.
19. The method of claim 18 , wherein the sub-optimal amount of platelet aggregation is less than 70% platelet aggregation.
20. The method of claim 18 , wherein a titration is first performed on the platelets to determine the amount of ADP suitable to induce a sub-optimal amount of platelet aggregation in the sample.
21. The method of claim 17 , wherein the ADP is present in the sample at a final concentration of 1 μM.
22. The method of claim 17 , wherein the ratio of hu5c8 antibody or antigen-binding fragment thereof to cross-linking agent is 1:1000 to 100:1, 1:500 to 500:1, or 3:2.
23. The method of claim 17 , wherein the sample comprising platelets is obtained from platelet rich plasma.
24. The method of claim 17 , wherein the hu5c8 antibody or antigen binding fragment thereof and the cross-linking agent are mixed prior to addition to the sample comprising platelets.
25. The method of claim 24 , wherein the hu5c8 antibody or antigen binding fragment thereof and the cross-linking agent are mixed for no less than 20 minutes prior to addition to the sample comprising platelets.
26. The method of claim 17 , wherein the hu5c8 antibody or antigen binding fragment thereof and the cross-linking agent are not a performed immune complex.
27. The method of claim 17 , wherein the amount of platelet aggregation in the sample is compared to the amount of platelet aggregation in a control sample.
28. The method of claim 27 , wherein the control sample is contacted with an aglycosylated hu5c8 antibody or antigen-binding fragment thereof or with a control human IgG.