Figure 9.

Formation and decay of C3bB(Mn²⁺) C3 proconvertase in the absence or in the presence of C3NeF IgGs, by microplate/WB assay. (A) Time course of C3bB(Mn²⁺) C3 proconvertase formation. The complexes were obtained incubating C3b-coated wells at 37°C for 1, 2, 4, and 8 h with 1,000 ng/ml FB and 2 mM MnCl₂ in the absence (-) or in the presence (+) of 2,640 ng/ml FH. The amount of C3bB formed was calculated as the density of B band (93 KDa), and reported in the bottom graph as Pixel²* 10⁶. (B–D) Time course of C3bB(Mn²⁺) C3 proconvertase spontaneous and FH-mediated decay. C3bB(Mn²⁺) complexes formed in 2 h at 37°C in the absence (B) or in the presence of C3NeF IgGs purified from patients P5 (C) and P10 (D), were further incubated with buffer alone (decay –) or with buffer added with 2,640 ng/ml FH (decay +), respectively, for 30, 60, 120, and 240 min at 37°C The percentage of residual B band was calculated as the ratio of the densities (in Pixel²) of each B band after decay and the corresponding baseline B band density before decay x 100 and results are reported in the bottom graphs. The membranes were then incubated with an anti-FH antibody and FH band could be visualized at 150 KDa (top). Results of a representative microplate/WB experiment of n = 3 are shown.