Effect of incubation order of rhFVIII, antibodies, and VWF on the prevention of C1q and CD32 binding. Binding to complement component C1q (left panels) and to CD32 (right panels) of rhFVIII (3 IU/mL), antibodies (0.5 μg/mL), and VWF (20 IU/mL) incubated in the indicated order was assessed by ELISA. As a source of anti-FVIII antibodies, either single anti-FVIII IgG Abs (Ab1 and Ab2), a combination (Ab1-7), or pooled serum from immunized F8^−/− mice (Ab-poly) were used. Bar charts show the binding relative to FVIII-IC alone. The mean value ± SD of 3 independent experiments done in duplicates is shown (individual data point are depicted). Comparison between groups with VWF addition was performed using 1-way ANOVA followed by HSD test with the Bonferroni correction (only significant differences are shown; ∗P < .05).