Representative FVIII competition curves of FVIII-specific antibody samples used for affinity ELISA validation. Samples containing FVIII-specific antibodies were preincubated with different concentrations of FVIII in solution (FVIII [nM]) and subsequently tested for the presence of remaining free antibodies (ΔOD-blank). A nonlinear distribution of FVIII concentrations was used for optimal coverage of different competition behavior in individual samples. A monoclonal human FVIII-specific IgG1 antibody spiked into human plasma not containing any FVIII-binding antibodies (sample A; circles) requires lower FVIII concentrations for competition than a human plasma sample containing low-affinity FVIII-specific IgG1 antibodies (sample B; triangles). A mixture of samples A and B was used to mimic bimodal affinity distribution (sample C; diamonds).