Figure 3.

AP contributes to haemolysis upon IgG3‐induced complement activity. Biotinylated RBCs were incubated with IgG1 (a), IgG3 (b) or IgM (c) antibiotin antibodies, with or without anti‐FB or anti‐C1q to induce haemolysis. The addition of anti‐C1q was able to inhibit haemolysis as induced by IgG1, IgG3 and IgM almost completely. EC50 was calculated to compare control to anti‐FB. With IgG1‐induced activation (a), there was no inhibition by anti‐FB. In IgG3‐induced activation (b), anti‐FB reduced haemolysis at most concentrations and statistically significant increased the EC50. In IgM‐induced activation (c), anti‐FB did not significantly reduce haemolysis or increase the EC50. (d) Haemolysis as initiated by an anti‐Vel IgM antibody in a more sensitive system, adding a FH‐inhibiting antibody and bromelain‐treated RBCs. At high anti‐Vel concentrations, there was no effect of inhibiting the AP, but at lowest antibody concentrations, AP inhibition had a statistical significant effect. Blocking the CP inhibited haemolysis completely. (a–c) Haemolysis as induced by milliQ was set to 100%. (a–c) Mean + SD from n = 3, statistical significance tested for EC50 using the paired t‐test; (d) mean + range from n = 2, statistical significance tested using one‐way ANOVA with Dunnett's multiple comparisons test, compared with the serum only condition. * P < 0.05, ** P < 0.01.