Doellgast G J, Rothberger H
Anal Biochem. 1985 Jun;147(2):529-34. doi: 10.1016/0003-2697(85)90310-0.
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.
已开发出一种用于凝血酶的新型固相微量滴定板测定法，该方法利用结合在微量滴定板孔中的纤维蛋白原和溶液中的过氧化物酶 - 纤维蛋白原作为指示系统。当向混合物中加入少量凝血酶时，会形成过氧化物酶 - 纤维蛋白和板结合纤维蛋白，并且过氧化物酶 - 纤维蛋白会与板结合纤维蛋白结合。过氧化物酶 - 纤维蛋白的结合量与凝血酶浓度和孵育时间成正比。使用该测定法，在150微升样品中可检测到低至0.25纳克/毫升（0.006纳摩尔）浓度的凝血酶。在特异性抑制剂苯甲脒和D - 苯丙氨酰 - L - 脯氨酰 - L - 精氨酸氯甲基酮存在的情况下，凝血酶活性会降低，抑制剂的相对浓度与其亲和力和作用机制一致。酶联凝血测定法通常可作为传统“基于凝块”的凝血试验的一种高度灵敏且便捷的替代方法。
