EVALUATION OF THE FIBRIN BINDING PROFILE OF 2 ANTIFIBRIN MONOCLONAL-ANTIBODIES

Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to hu man freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to Var ious domains of the fibrinogen/fibrin moiety. Using plasmin-mediated f ibrinogen digests, immunoblots showed that both MAbs crossreacted stro ngly with fragments X and Y, weakly with fragment-E and not at all wit h fragment D, Purified fragments D and E used in an ELISA confirmed th at MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E . Surface Plasmon Resonance (SPR) technology, employed to further eval uate the epitopes in fibrin, showed that MAb 1H10 had a higher affinit y for fragment-E (K-D = 8.04 x 10(-9) M) than MAb 5F3 (K-D = 1.13 x 10 (-8) M). Individual association and dissociation rate constants of 7.9 7 x 10(5) M(-1) s(-1) and 3.97 x 10(-3) s(-1), respectively, for MAb 1 H10, and 5.16 x 10(5) M(-1) s(-1) and 3.62 x 10(-3) s(-1), respectivel y, for MAb 5F3 were also obtained, A SPR inhibition assay confirmed th at MAb 1H10 had a greater affinity for fragment-E than MAb 5F3, Howeve r individual isolated polypeptide chains of fibrinogen fragment E (E-A alpha, E-B beta, E-gamma) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures, Furthermore, SPR pair -wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E, These distinct epitopes appeare d to br a conformational amalgam of linear sequences in two or three o f the polypeptide chains of fragment-E, or distinct conformational epi topes on one of the three subunit chains alone.