Evaluation of the fibrin binding profile of two anti-fibrin monoclonal antibodies.
Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that 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 evaluate the epitopes in fibrin, showed that MAb 1H10 had a higher affinity for fragment-E (KD = 8.04 x 10(-9) M) than MAb 5F3 (KD = 1.13 x 10(-8) M). Individual association and dissociation rate constants of 7.97 x 10(5) M-1s-1 and 3.97 x 10(-3)s-1, respectively, for MAb 1HAb 1H10, and 5.16 x 10(5) M-1s-1 and 3.62 x 10(-3)s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However 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 appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.
Raut
S
S
Division of Haematology, National Institute for Biological Standards and Control, Potters Bar, Herts, UK.
Gaffney
P J
PJ
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005337:Fibrin / Q000276:immunology / Q000378:metabolism*; D005338:Fibrin Fibrinogen Degradation Products / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting
D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; C015214:fibrinogen D fragment; C018543:fibrinogen fragment E; D005337:Fibrin