프로트롬빈 두단계측정법에서 프로트롬빈을 트롬빈으로 전환시키는데 Echis carinatus독으로부터 분리된 프로트롬빈 활성화 효소인 프로트롬빈을 트롬빈으로 약 30분이내에 거의 전환시켰다. 생성된 트롬빈의 활성은 반응시간이 20시간 경과하여도 안정하게 유지되었다. 한편 프로트롬빈을 혈장-혈청 혼합물을 이용하여 트롬빈으로 전환시켰을 때는 약 9분정도 경과되면 생성된 트롬빈의 활성이 감소함을 보여주었다. 트롬빈 효소의 기질로써 피브리노젠과 합성핵원체 기질을사용하여 프로트롬빈을 측정할 수 있는 농도의 범위를 조사하였다. Clotting assay에서 프로트롬빈 측정 가능범위는 2 ${mu}g/ml$-100 ${mu}g/ml$이었으며 chromogenic assay에서는 0.2 ${mu}g/ml$-16 ${mu}g/ml$이었다. 프로트롬빈을 활성화시키지 않고 직접 측정하기위하여 효소를 이용한 면역측정법 (ELlSA)을 개발하였다. lndirect ELlSA방법에 의해서는 최소 40 ng/ml, sandwich ELlSA를 사용하여 소 혈장의 프로트롬빈 농도를 측정 한 결과 224 ng/ml이었으며 within-run은 9.3%, between-run은 15.8%의 변화를 보여주었다.
In a two-stage assay of prothrombin, prothrombin was activated to thrombin by ecarin, the prothrombin activator from the venom of Echis carinatus, in the absence of calcium ions and phospholipid. The activation was almost complete within 30 minutes and the thrombin generated was not inhibited by the ecarin activation mixtures for a incubation time up to at least 20 hours, while it was inhibited by the absorbed plasma- serum mixture after 9-minute incubation. Two different substrates, fibrinogen and a synthetic chromogenic substrate, were used for assays of the thrombin activity and compared in their sensitivies in detection of prothrombin. The standard curve was linear between 2 ${mu}g/ml$ of prothrombin concentration in the clotting assay, and between 0.2 ${mu}g/ml$ and 16 ${mu}g/ml$ in the chromogenic assay. In addition we developed the enzyme-linked-immunosorbent assays for the direct measurement of prothrombin. The minimum detectable concentrations of prothrombin by the indirect ELISA and the sandwich ELISA were 40 ng/ml and 3 ng/ml, respectively. The prothrombin level in bovine plasma was determined to be 224 ng/ml by the sandwich ELISA. The reliability of the sandwich ELISA was demonstrated by the within runs and between runs tests, which were 9.3% and 15.8%, respectively.
