A Monoclonal Antibody Specific for Two-Chain Urokinase-Type Plasminogen Activator. Application to the Study of the Mechanism of Clot Lysis With Single-Chain Urokinase-Type Plasminogen Activator in Plasma

A murine monoclonal antibody (MA-1 2E6A8) was raised against human urokinase-type plasminogen activator (uPA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant twochain U-PA (rtcu-PA) than with recombinant single-chain U-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA-l2E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated U-PA specific antibody for tagging. It recognized free and active site blocked rtcu-PA but not rtcu-PA in complex with rPAI-1 or with a,-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 fig/mL rscu-PA to 3 mL plasma containing a 0.2 mL ‘261-fibrin