Platelet aggregation assays using a CD40L-binding agent
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. A method of assaying platelet aggregation comprising:
contacting platelets with a platelet activating agent;
contacting the activated platelets with a CD40L-binding moiety; and
contacting the activated platelets with a cross-linking agent, wherein sedimented platelets is indicative of aggregation of the platelets.
2. The method of claim 1 wherein the platelet activating agent is selected from ADP, collagen, thrombin, thromboxane, neutrophil elastase, p-selectin, and convulxin.
3. The method of claim 1 wherein the platelet activating agent is ADP.
4. The method of claim 1 wherein the platelets are obtained from platelet rich plasma.
5. The method of claim 1 wherein the CD40L-binding moiety is an anti-CD40L antibody.
6. The method of claim 5 wherein the anti-CD40L antibody is hu5c8.
7. The method of claim 1 wherein the cross-linking agent is selected from sCD40L, anti-human IgG antibody, anti-hFc antibody, RF, Fc receptor-positive accessory cell, soluble protein A, and soluble human Fc receptor.
8. The method of claim 1 wherein the cross-linking agent is selected from sCD40L, anti-human IgG antibody, and anti-hFc antibody.
9. The method of claim 1 wherein the ratio of CD40L-binding moiety to cross-linking agent is 1:1000 to 1000:1.
10. The method of claim 1 wherein the ratio of CD40L-binding moiety to cross-linking agent is 1:500 to 500:1.
11. The method of claim 1 wherein the ratio of CD40L-binding moiety to cross-linking agent is 3:2.
12. A method of assaying platelet aggregation comprising:
activating platelets with ADP;
contacting the activated platelets with an anti-CD40L antibody; and
contacting the activated platelets with sCD40L or anti-hFc antibody, and wherein sedimented platelets is indicative of aggregation of the platelets.
13. A method of determining whether a human is susceptible to clotting upon administration of a CD40L-binding moiety comprising:
removing platelets from the human;
contacting the platelets with a platelet activating agent;
contacting the activated platelets with the CD40L-binding moiety; and
contacting the activated platelets with a cross-linking agent, and
determining the presence or absence of platelet aggregation, wherein 70% or greater platelet aggregation is indicative of susceptibility to clotting.
14. The method of claim 13 wherein the platelet activating agent is selected from ADP, collagen, thrombin, thromboxane, neutrophil elastase, p-selectin, and convulxin.
15. The method of claim 13 wherein the platelet activating agent is ADP.
16. The method of claim 13 wherein the platelets are obtained from platelet rich plasma.
17. The method of claim 13 wherein the CD40L-binding moiety is an anti-CD40L antibody.
18. The method of claim 17 wherein the anti-CD40L antibody is hu5c8.
19. The method of claim 13 wherein the cross-linking agent is selected from sCD40L, anti-human IgG antibody, anti-hFc antibody, RF, Fc receptor-positive accessory cell, soluble protein A, and soluble human Fc receptor.
20. The method of claim 13 wherein the cross-linking agent is selected from sCD40L, anti-human IgG antibody, and anti-hFc antibody.
21. The method of claim 13 wherein the ratio of CD40L-binding moiety to cross-linking agent is 1:1000 to 1000:1.
22. The method of claim 13 wherein the ratio of CD40L-binding moiety to cross-linking agent is 1:500 to 500:1.
23. The method of claim 13 wherein the ratio of CD40L-binding moiety to cross-linking agent is 3:2.
24. A method of determining whether a human is susceptible to clotting upon administration of an anti-CD40L antibody comprising:
removing platelets from the human;
activating the platelets with ADP;
contacting the activated platelets with the anti-CD40L antibody; and
contacting the activated platelets with sCD40L or anti-hFc antibody, and determining the presence or absence of platelet aggregation, wherein 70% or greater platelet aggregation is indicative of susceptibility to clotting.
25. The method of claim 1 , wherein the CD40L-binding moiety and the cross-linking agent are not a preformed immune complex.
26. The method of claim 12 , wherein the anti-CD40L antibody and the sCD40L are not a preformed immune complex.
27. The method of claim 13 , wherein the CD40L-binding moiety and the cross-linking agent are not a preformed immune complex.
28. The method of claim 24 , wherein the anti-CD40L antibody and the sCD40L are not a preformed immune complex.