Figure 3.

Assessing FI function. In the presence of a cofactor (A) FH 50 nM; (B) sCR1 25nM; (C) MCP 50nM, C3b cleavage activity by FI in patient serum (diluted 1:32) is measured using C3b-decorated sheep erythrocytes. Non-cleaved C3b is developed by the addition of both FB/FD to form C3 convertase and rat-EDTA serum as a source of C5-C9. Percent hemolysis is calculated and plotted as a function of FI concentration determined by ELISA. The grey line in each figure serves as a normal reference line calculated from a normal sample (FI concentration, 40 mg/L) with serial dilutions. Serum from a CFI c.1429+1G>C homozygote (FI is undetectable) serves as a positive control (black dot). (D) Circulating FI by a Western blot. Patient sample (1:80 diluted in PBS) with reducing reagent separated on a 4-15% polyacrylamide gel and transferred. FI was visualized by an antibody specific to the heavy chain (50k Da). CFI p.Arg336Gly results in an unprocessed single chain (88k Da).