SELECTION OF MONOCLONAL-ANTIBODIES THAT BIND AND INHIBIT TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Three monoclonal antibodies raised against tissue-type plasminogen act ivator (t-PA) were selected for their ability to inhibit solid-phase b ound t-PA. Each monoclonal antibody blocked the release of p-nitroanil ine from H-D-Ile-Pro-Arg-pNA (S-2288). The first antibody 1D2 was a ga mma2b,kappa with K(D) = 8 x 10(-9) M, the second antibody 2B9 was a ga mma1,kappa with K(D) = 2 x 10(-9) M, and the third antibody 5A9 was a gamma1,kappa With K(D) = 4 x 10(-10) M. In solution-phase format each antibody blocked the conversion of plasminogen to plasmin as judged by a plasmin assay and also inhibited t-PA-mediated lysis of plasma fibr in clot in plasma. The binding of each I-125-radiolabeled antibody to t-PA was inhibited by any one of the three antibodies, suggesting that they recognized a common epitope on t-PA which was absent on unfolded t-PA. We concluded these antibodies bind near t-PA active site since PPACK treatment lowered binding of two antibodies. We believe solid-ph ase chromogenic substrate assay may be a useful way to screen for anti bodies directed against the active site of proteases.