FIGURE 3.

Analysis of peripheral blood plasma of mice receiving Fc.Mut24 or PBS. Peripheral blood was collected via retro-orbital bleeding in sodium citrate and spun at 500g for 5 min to separate plasma from cells. (A) ELISA was performed to measure anti-FVIII antibody levels in peripheral blood plasma using mouse whole IgG as control. p = 0.021 between Fc.Mut24-treated (n = 8) and PBS-treated (n = 4) groups. (B) Bethesda assay was performed on peripheral blood plasma. Serial dilutions were performed on plasma samples that showed high levels of inhibitor to obtain accurate inhibitor titers. p = 0.0092 between Fc.Mut24- and PBS-treated groups at multiple time points. (C) Factor VIII activity was measured in peripheral blood plasma via activated partial thromboplastin time assay relative to normal human plasma controls. p = 0.0014 between Fc.Mut24- and PBS-treated groups at multiple time points. The experiments were repeated twice with no significant variation. The data are presented as means with standard deviation. The p-values were calculated for multiple time points between groups using repeated-measures ANOVA.