Measurement of plasminogen activator inhibitor 1 (PAI-1) in plasma with various monoclonal antibody-based enzyme-linked immunosorbent assays

Abstract

Four monoclonal antibodies, MA-7D4, MA-7F5, MA-12A4 and MA-15H12, were raised against human plasminogen activator inhibitor-1 (PAI-1). MA-7D4 and MA-7F5 had an inhibitory effect on PAI-1 activity, whereas MA-15H12 and MA-12A4 did not affect PAI-1 activity. MA-7D4 was used previously as capture antibody in combination with MA-7F5 conjugated to horseradish peroxidase (7F5-HRP) to construct an ELISA which was 12 times more sensitive towards free PAI-1 as compared to PAI-1 in complex with human tissue-type plasminogen activator (t-PA) (Blood 71: 220–225, 1988). MA-15H12, as capture antibody, in combination with MA-12A4 corjugated to horseradish peroxidase (12A4-HRP) yielded an ELISA which was equally sensitive towards free PAI-1 and PAI-1/t-PA complex. Another ELISA was constructed using MA-15H12 as capture antibody in combination with an anti-t-PA antibody (MA-62E8) conjugated to horseradish peroxidase (62E8-HRP), allowing the specific measurement of t-PA/PAI-1 complexes and the development of an immunofunctional method for the specific quantitation of active PAI-1. These assays were then used to measure PAI-1 levels in plasma and the values obtained were compared with the data obtained by functional methods. It was found that 44% to 60% of free PAI-1 antigen in normal plasma is active and that t-PA occurs mainly as t-PA/PAI-1 complexes. In plasma from 48 patients suffering from angina pectoris no statistical difference in PAI-1 activity. PAI-1 antigen or PAI-1/t-PA complex could be observed as compared to normal subjects. The present assays are based on stable and reproducible reagents and allow the specific determination of free PAI-1 antigen, total PAI-1 antigen, PAI-1 activity and t-PA/PAI-1 complexes. They may facilitate further investigation of the role of PAI-1 in clinical conditions associated with impaired fibrinolysis.