EPITOPE MAPPING OF HUMAN FACTOR-IX INHIBITOR ANTIBODIES

We have determined the location of epitopes on the factor M for three haemophilia B inhibitor antibodies (HB-1, HB-3, HB-7) and a monoclonal anti-factor TX inhibitory antibody (designated 65-10). The main binding region of HB-1, HB-3 and HB-7 was (155)YVNSTEAETI(164) (residues 155-164), (167)NITQSTQSFN(176) and (156)VNSTEAETTI(164), respectively. The binding region of 65-10 was (168)ITQSTQSFNDFTRVV(182), which included the cleavage site ((180)R-V-181) for activation by factor XIa. By neutralization experiments using two peptides. (156)VNSTEAETI(164) and (167)NITQSTQSFN(176), the degree of neutralization of anti-factor TX IgG purified by protein A was determined. Neutralization of three antibodies, HB-1, HB-3 and HB-7, in the presence of 10mM of the peptides (156)VNSTEAETI(164) was 30.1%, 0% and 10.8%, respectively, and in the presence of 4 mM of (167)NITQSTQSFN(176) it was 0%, 13.5% and 17.3%, respectively. On the other hand, when plasmas of patients instead of purified IgG were used for neutralization, 10 mM of (156)VNSTEAETI(164) and 4 mM of (167)NITQSTQSFN(176) failed to neutralize the inhibitor in the plasmas.