CHARACTERIZATION OF THE CHAINS OF HUMAN FIBRINOGEN ISOLATED BY PERFUSION CHROMATOGRAPHY USING FIBRIN SPECIFIC MONOCLONAL-ANTIBODIES

In order to study the epitopes on fibrin to which monoclonal antibodie s are directed, we required pure individual polypeptide chains of huma n fibrinogen in milligram quantities. High purity chains of human fibr inogen were rapidly obtained, in under 3 minutes, by the novel procedu re of reversed-phase perfusion chromatography and these chains were su bjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these anti bodies in both immunoblotting and enzyme linked immunospecific assay ( ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were emp loyed to initiate studies to define the epitopes in fibrin to which fo ur fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are t argeted. Two of these antibodies, B10 and A11, were shown to be direct ed to a linear sequence on the Aa chain, although the binding profiles for the two antibodies suggested that different epitopes may be invol ved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that confo rmational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.