Journal: Clinical and Experimental Immunology
Article Title: A novel fusion protein reduces kidney complement in experimental C3 glomerulopathy
doi: 10.1093/cei/uxag015
Figure Lengend Snippet: FHR5 1-9 FH 1-5 binds to C3, destabilizes C3 convertase and acts as a cofactor for factor I. Binding profiles of increasing amounts of FHR5 1-9 FH 1-5 to surface bound C3, C3b, iC3b, C3d and BSA (negative control), detected with both anti-CFHR5 (a) and anti-FH (b) antibodies. Data shown are mean values of triplicate measurements; error bars denote the standard deviation. C3a generated by convertase formation on surface-bound C3b: ELISA plates were coated with purified C3b. Surface C3 convertase was formed by addition of purified factor B (FB), factor D (FD), and properdin (P). Then increasing amounts of either FH (c) or the FHR5 1-9 FH 1-5 (d) were added followed by addition of C3 to enable C3a generation. To investigate factor I (FI) co-factor activity, FI along with increasing amounts of either FH (e) or the FHR5 1-9 FH 1-5 (f) were added prior to the convertase formation step. NC—negative control, no FB, FD, or P added. PC—positive control, no FH, or FHR5 1-9 FH 1-5 added. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test; **** P < 0.0001. Fluid phase co-factor assay products were visualized by western blotting (g). Cofactor activity was indicated by the cleavage of C3b as determined by the presence of C3 α-chain fragments (arrow). Negative controls: C3, C3b, C3b with FH, C3b with FI, and FHR5 1-9 FH 1-5 alone. Positive control: C3b incubated with FH and FI.
Article Snippet: After incubation and washing, bound FHR5 1-9 FH 1-5 was detected using goat polyclonal anti-CFHR5 antibody (R&D systems), followed by mouse anti-goat/sheep IgG-HRP (Sigma) and finally TMB substrate (BD).
Techniques: Binding Assay, Negative Control, Standard Deviation, Generated, Enzyme-linked Immunosorbent Assay, Purification, Activity Assay, Positive Control, Western Blot, Incubation
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