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 μl 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. © 1985.
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