DETECTION OF SPECIFIC FORMS OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1-BY MONOCLONAL-ANTIBODIES

Monoclonal antibodies to plasminogen activator inhibitor type-1 (PAI-1) were generated and characterised for their ability to inhibit PAI-1 interaction with tissue plasminogen activator (t-PA) and urokinase (u-PA) and detection of the various forms of PAI-1 (native, complexed, and degraded) by immunoblotting. Mab17 inhibited both complex formation between PAI-1 and t-PA/u-PA and PAI-1 activity in a dose dependent manner by 90%. Mab 25 was much less effective, blocking complex formation less than 30% and PAI-1 activity less than 20%. The Kds of Mab17 and Mab25 were 2.8 X 10(-11) M and 2.6 X 10(-10) M, respectively. Following SDS-PAGE and immunoblotting, Mab17 detected native PAI-1 only; PAI-1 in complex and the t-PA/u-PA degraded form of PAI-1 (M(r) = 42000) did not react with this antibody. In contrast, Mab25 detected all three forms of PAI-1 although the affinity for the native form appeared to be greater than Mab17 or the PAI-1 polyclonal employed. Despite these differences, both monoclonal antibodies immunoprecipitated native and degraded PAI-1 equally as well. These results suggest that the epitope of Mab17 is associated with the reactive site of PAI-1 and that this region is either missing or not accessible in the cleaved form or in complex.