Differential detection of single chain urokinase-type plasminogen activator and two chain urokinase-type plasminogen activator in human and monkey plasma

Abstract An ELISA-based assay system is described for the selective measurement of recombinant single chain (rscu-PA) and two chain urokinase-type plasminogen activator (rtcu-PA) when present together in human or in monkey plasma. The assay system consists of two separate sandwich ELISAs, one for rscu-PA which utilises two monoclonal antibodies, and another one for total u-PA, which utilises a polyclonal antibody both as capture and as tagging antibody. Since the latter was found to be less sensitive to rtcu—PA-inhibitor complexes than to uncomplexed rscu-PA in plasma, plasmin activation of rscu-PA was introduced in order to eliminate this variable. Complete inhibitor complex formation was shown to occur after incubation (⩾2h). Excess inhibitors were provided by making dilutions with normal plasma. The assay detection limit, in plasma, for total u-PA antigen and rscu-PA was 0.8 and 3 ng/ml, respectively. The rtcu-PA concentration was calculated by subtracting the rscu-PA from the total u-PA concentration. The assay system was used to analyse blood samples from Cynomolgus monkeys infused with 90000 IU (0.6 mg)/kg of rscu-PA infused over 1h. By the end of the infusion, the rtcu-PA concentration was 0.5 μ/ml, whereas that of rscu-PA was 1 μg/ml. No fibrinogenolysis occurred. However, when the rscu-PA infusion was preceded by a bolus of 400000 IU (0.8 mg)/kg of rt-PA, rtcu-PA generated reached 1.35 μg/ml with 30% fibrinogenolysis. The ELISA system described can be used to provide a better understanding of the relationship between systemic scu-PA activation and thrombolytic activity, fibrin specificity and the co-use of other agents such as t-PA or tcu-PA.