Indirect epitope mapping of anti-C1 IgG in patients with hemophilia by competition binding with mAb 2A9. Binding of patient IgG to the human C1 domain presented as hC1pfVIII protein (A) and HSA-hC1 domain protein (B and C) was analyzed in the absence (pink bars) and presence (red bars) of mAb 2A9. Purified hC1pfVIII (A) and HSA-hC1 (B-C) proteins were immobilized on microtiter plates, and antibody binding from patient plasma was detected with an HRP-conjugated goat anti-human IgG. Antibody binding to immobilized pfVIII (A) and HSA alone (B and C) was subtracted to specifically analyze binding of non-cross-reactive (A) and cross-reactive and non-cross-reactive (B-C) anti-C1 IgG in patient plasma. The experiments were repeated twice with similar results. (D) Comparison of patients with AHA (B; n = 15) and patients with HA and inhibitors (C; n = 10) revealed a significant difference regarding the inhibition of IgG binding to HSA-hC1 by mAb 2A9 (P = .0009). Data are presented as median with interquartile range (*P < .05; **P < .01; ***P < .001. Mann-Whitney test).