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

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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.