An enzyme-linked immunosorbent assay (ELISA) for the measurement of plasmin-alpha 2-antiplasmin complex in human plasma--application to the detection of in vivo activation of the fibrinolytic system

Abstract

An enzyme-linked immunosorbent assay (ELISA) is developed for the measurement of plasmin-alpha 2-antiplasmin complex in human plasma. Microtiter plates were coated with a mixture of two murine monoclonal antibodies directed against human alpha 2-antiplasmin and bound plasmin-alpha 2-antiplasmin complex was quantitated with a peroxidase-conjugated monoclonal antibody directed against human plasminogen. The lower limit of sensitivity of the assay was 0.01 nM of plasmin-alpha 2-antiplasmin complex in 100-fold diluted human plasma, allowing detection of 1 nM in undiluted plasma samples. After 100-fold dilution of the plasma samples, the assay was no longer influenced by the presence of the precursors plasminogen and alpha 2-antiplasmin. At a concentration of 2.0 nM of plasmin-alpha 2-antiplasmin complex in plasma, intra- and interassay variation coefficients were 4.2 and 5.5 percent respectively. In plasma samples of 25 control subjects the levels of plasmin-alpha 2-antiplasmin complex were below 1 nM. Extensive in vivo activation of the fibrinolytic system during thrombolytic therapy with streptokinase resulted in the generation of elevated levels of plasmin-alpha 2-antiplasmin complex up to 690 +/- 150 nM. No measurable levels of plasmin-alpha 2-antiplasmin complex were found in the plasma of 32 patients with acute deep vein thrombosis nor in the plasma of 11 patients with recurrent deep vein thrombosis. These findings indicate that plasmin-alpha 2-antiplasmin complex is generated during in vivo activation of the fibrinolytic system and that its assay may be useful to monitor thrombolytic therapy but not for the diagnosis of venous thrombosis.