A method for the determination of the affinity constant of antibodies to tissue plasminogen activator

A simple method for the determination of the affinity constant of antibodies to fibrin-bound tissue-type plasminogen activator (t-PA) is described. The method is based on the high-affinity binding of t-PA to a solid-phase fibrin support which allows the specific separation of t-PA present in any biological fluid. This provides as in the physiological fibrin-t-PA interaction, a molecular structure conserving its antigenic and functional properties which allows for the binding of specific anti-t-PA antibodies and hence facilitates the separation of free from bound antibody. Then, antibodies bound to t-PA are revealed with a labelled, second antibody and data relating Bound v Total antibody input can be analyzed. Affinities were calculated by least square nonlinear regression analysis using a computerized approach. The method allows the direct use of plasma or other crude sources where t-PA is present in its most native form.