Devepolment of a high sensitivity assay for activated coagulation fadctor-inhibitor complex and its diagnostic application to thrombosis

采用活化巯基- sepharose建立了一种酶联免疫吸附法（ELISA），用于定量活化的人因子ix -抗凝血酶III （IXa-AT III）复合物。IXa-AT III复合物与巯基- sepharose小柱结合，偶联抗AT III的单特异性兔抗体。然后用第二抗体rabbit anti-human factor IX Fab'定量IXa-AT III复合物，标记为< β >-d-半乳糖苷酶。将终浓度为5.0 ng[1.0 ug/ml]的纯化IXa-AT III复合物加入缓冲液或血浆中，检测IXa-AT III复合物。该复合物的最低可检测量（5 ng/ml）相当于激活血浆因子IX的0.05%。用纯化的F.IXa、AT III、正常血浆和AT III耗尽血浆进行的实验证明了该方法的特异性。我们测量了在玻璃管中凝血正常血液中Ixa-At III复合物的生成，发现Ixa-At III复合物在静脉穿刺后60秒内出现。10例正常人复合物患者的天然全血中复合物的含量均未低于5.0 ng/ml，而3例弥散性血管内凝血（DIC）患者的复合物含量均超过20 ng/ml。本文描述的使用活化巯基- sepharose的ELISA似乎提供了一种有用的工具来检测体外和体内凝血系统的激活。

An enzyme-linked immunosorbent Assay (ELISA) using activated Thiol-Sepharose has been developed for the quantitation of activated human factor IX-antithrombin III (IXa-AT III) complex. IXa-AT III complex is bound to a mini-column of thiol-Sepharose to whixh monospecific rabbit antibody against AT III was coupled. IXa-AT III complex is then quantified by a second antibody, rabbit anti-human factor IX Fab', labeled with <beta>-d-galactosidase. IXa-AT III complex was detected, when a final concentration of 5.0 ng[1.0 ug/ml of purified IXa-AT III complex was added to buffer or plasma. The minimum detectable amount of the complex (5 ng/ml) corresponds to activation of as little as 0.05% of plasma factor IX. Experiments with purified F.IXa, AT III, normal plasma and AT III-depleted plasma demonstrated the specificity of the assay. We measured Ixa-At III complex generation in clotting normal blood in glass tubes, and found that IXa-AT III complex appears within 60 sec after venipuncture. No detectable amount of the complex was observed in native whole blood from patients complex in 10 normal individuals were less than 5.0 ng/ml, wheresa those in 3 patients with disseminated in travascular coagulation (DIC) were more than 20 ng/ml. The ELISA using activated thiol-Sepharose described in this paper seems to provide a useful tool to detect activation of coagulation system in vitro as well as in vivo.