Figure 5.

Effect of properdin on C3 convertase-stabilizing activity of C3NeF IgGs. (A) Experimental design (protocol 3). (B,C) Representative images of the assay. C3bBb(Mg²⁺) complexes were formed by incubating at 25°C for 12 min C3b-coated wells with 1,000 ng/ml FB, 10 ng/ml FD, 100 μg/ml IgGs from patients or healthy controls and 10 mM MgCl₂ in the presence of 500 ng/ml Properdin (P) (baseline). In additional wells after washing, spontaneous or FH-mediated decay of the formed complexes was monitored by further incubation for 32 min at 25°C with buffer alone (decay –) or buffer added with 2,640 ng/ml FH (decay +), respectively. The percentage of residual Bb band was calculated as the ratio of the densities (in Pixel²) of each Bb band (visualized by an anti-FB antibody) after decay and the corresponding baseline Bb 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 for each sample are shown.