Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin.
A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time of incubation. Using this assay, thrombin was measured at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliter of sample. In the presence of the specific inhibitors benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at relative concentrations of inhibitors consistent with their affinities and mechanisms of action. The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient alternative to conventional "clot-based" tests of coagulation.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001550:Benzamidines / Q000494:pharmacology; D001777:Blood Coagulation; D005337:Fibrin; D006801:Humans; D008722:Methods; D010544:Peroxidases; D013917:Thrombin / Q000032:analysis*
D000590:Amino Acid Chloromethyl Ketones; D001550:Benzamidines; D005337:Fibrin; D010544:Peroxidases; D013917:Thrombin; C032157:benzamidine; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone