Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B-M by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulatio n factor IX (FIX), designated 65-10, which interfered with the activation o f FIX by the activated factor XI/Ca2+ and neutralized the prolonged ox brai n prothrombin time of hemophilia B-M [11,12]. The location of the epitope o n the FIX for 65-10 MoAb is (168)Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Ph e-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an ep itope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope pepti de on the prolonged ox brain prothrombin time of the hemophilia B-M plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, V al, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178)Phe --> Pro. These results imply that a hydrophobic molecule of (1 75)Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutrali zed the prolonged ox brain prothrombin time of hemophilia B-M Nagoya 2 ((18 0)Arg --> Trp) and Kashihara ((181)Val --> Phe) as well as B-M Kiryu ((313) Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168)Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr- Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitat ive analysis of hemophilia B-M. (C) 2000 Elsevier Science Inc. All rights r eserved.