Inhibitory anti-fVIII antibodies in hemophilia A mice immunized with HSQ, HSQ R484A/R489A/P492A, or HSQ R484A/R489A. Hemophilia A mice received 6 intravenous injections of 10 μg/kg HSQ, HSQ R484A/R489A/P492A, or HSQ R484A/R489A at 14-day intervals, followed by a final injection of 25 μg/kg 2 weeks after the sixth dose. Blood samples were taken 2 weeks after the final injection and were tested for inhibitory anti-fVIII antibodies by a modified Bethesda assay using isologous antigen in human hemophilia A plasma as substrate, as described in “Materials and methods.” Horizontal lines represent the sample means. Statistical parameters of the samples (mean ± SD) were 670 ± 500, 320 ± 310, and 780 ± 570 for the HSQ, HSQ R484A/R489A/P492A, and HSQ R484A/R489A groups, respectively.