Application of enzyme immunoassays for testing haemocompatibility of biomedical polymers

Abstract

In this study enzyme immunoassays are presented for the assessment of platelet adhesion/activation and fibrinogen adsorption/conformation. The estimation of platelet adhesion and activation was performed with two enzyme immunoassays (EIAs) using monoclonal antibodies directed against CD42b (GP Ib) and CD 62 (GMP 140 or P-Selectin). The applicability of EIA was first demonstrated in microtitre plates coated with fibrinogen. The thrombogenic substrate showed that platelet adhesion and activation reached a plateau level within 30 min. The use of EIA for testing biomaterials was demonstrated with polymeric reference materials where a differentiation of materials with respect to adhesion and activation was achieved. To validate the EIA scanning electron microscopy was applied and confirmed the different extent of adhesion and activation of platelets on reference materials. In addition, polyurethaneureas, based on 4,4′-diphenylmethane diisocyanate and polytetramethylene glycols, with different hard segment content and composition were investigated. It was found that both adhesion and activation were not simply dependent on the hard segment content but also on the hard segment composition. To get more insight into the mechanism of this process, two EIAs for the binding of fibrinogen using polyclonal and monoclonal antibodies were developed. There it was shown that the adhesion and activation of platelets on polyurethaneureas was not simply dependent on the total amount of adsorbed fibrinogen but rather on its conformation, indicated by the binding of the monoclonal antibody directed vs the γ-chain of fibrinogen.