NOVEL ACTIVITY AND IMMUNO - ASSAYS FOR PAI-1

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Plasminogen activation is modulated by a number of inhibitors. he fast acting inhibitor of t-PA and u-PA has been designated plasminogen activator inhibitor-1 )PAI-1). The detection of raised levels of this acute phase protein is indicative of an imbalance in the hemostatic system. In order to fully assess PAI-1 plasma levels it is necessary to measure the protein by both antigenic and activity assays. We propose an approach which will allow the measurement of total plasma antigen levels, PAI-1/t-PA antigen levels, PAI-1 activity levels, and the activity and antigen levels of vitronectin/PAI-1 complex which represents a special, stable subpopulation of PAI-1 which may have antithrombin activity. The activity assay involves the use of an enzyme labelled fibrin matrix as the substrate for t-PA; residual activity will allow the measurement of PAI-1 inhibitory activity. We propose to use both standard ELISA assay and the ultrasensitive ELISA-ELCA assay technique. This is because levels of free PAI-1 antigen and complexed PAI-1 differ by at least 10 fold. With the preparation of reagents to carry out these assays we will be able to do initial tests on assays for plasma components such as the vitronectin/PAI-1 complexes and u-PA PAI-1 complexes.