Human factor b

To elucidate the effect of CTRP6 on AP C3 convertase activity, 1 μg human C3 (Sigma), 1 μg human factor B (CompTech, USA), 100 ng human factor D (CompTech) and 0–100 ng rhCTRP6 (Aviscera Bioscience) in 15 μl of reaction buffer (2 mM CaCl₂, 0.5 mM MgCl₂, 20 mM Tris-HCl (pH 7.6)) were incubated at 37 °C for 1 h. The samples were subjected to SDS–PAGE and visualized by CBB staining. The inhibitory effect of CTRP6 on AP C3 convertase activity was calculated from the intensity of factor B protein band, measured using ImageJ. To investigate the association between CTRP6 and factor H, 1 μg C3, 1 μg factor B and 100 ng factor D with/without 100 ng rhCTRP6 and/or 1 μg factor H (CompTech) in 15 μl of reaction buffer were incubated at 37 °C for 1 h, and the reaction mixtures were subjected to SDS–PAGE and visualized by CBB staining.

In the case where the polyreactivity of human monoclonal IgG1 was assessed, the microtiter plates were coated with a panel of antigens: human factor IX (LFB); human factor VIII (Kogenate FS, Bayer HealthCare); porcine tubulin (Sigma-Aldrich), human C-reactive proteins (Calbiochem, San Diego, CA, USA), human factor B (CompTech, Tyler, TX, USA) human hemoglobin (Sigma-Aldrich); human VWF (Wilfactine, LFB), bovine myelin basic protein (Sigma-Aldrich); porcine thyroglobulin (Sigma-Aldrich); diphtheria toxoid and lipopolysaccharides from Escherichia coli O55:B5 (Sigma-Aldrich). This panel was selected to represents diverse and unrelated autoantigens and foreign antigens. Ab21 at 5 µM was exposed to 1 µM of metHb for 1 hour at ice. After the antibody was diluted 10× in T-PBS and incubated with the immobilized proteins for 2 h at room temperature. Next steps of the experiment are identical as those described above.