Detection of specific proenzyme activators in snake venoms by a new immunoabsorbant-chromogenic substrate method

In separate experiments, antibodies to plasminogen, factor X and protein C were applied to microtitre trays as commonly used in enzyme-linked immunoassays. After incubation with dilute normal human plasma as a source of the corresponding proenzyme antigen, the wells were exposed to dilutions of various snake venoms. After thorough washing, the microtitre tray wells were tested overnight with chromogenic tripeptide substrates known to be relatively specific for the activated forms of the above factors, i.e., plasmin, factor Xa and activated protein C. The immunochromometric assay described detected two new activators of protein C in Agkistrodon piscivorus and Agkistrodon contortrix venoms and a new factor X activator in Agkistrodon rhodostoma venom. Gel filtration of the latter venom indicated that the factor X activator eluted with high molecular weight, was clearly distinct from the peak fibrinogen clotting activity (Ancrod) and appeared to have no procoagulant activity. Although several Bothrops venoms appeared to contain plasminogen activator by this technique, the observed strong chromogenic activity was observed in microtitre wells independently of plasminogen and represented nonspecific amidase activity.