Solid Phase Convertase Formation and Analysis

The solid phase AP C3 convertase was built up as described by Hourcade et al. in 2002 (37 (link)) with modifications. C3b was immobilized at 5 µg/ml on microplate wells (Maxisorp, NUNC). After washing, 2 µg/ml FB, 4 µg/ml properdin and 0.1 µg/ml FD were added in convertase buffer (DPBS containing 4% BSA, 2 mM NiCl₂, 0.1% Tween-20) for 1 hour at 37°C. Convertase formation was detected with goat anti-FB and HRP-conjugated rabbit anti-goat antibodies. Convertase activity was determined by adding 10 µg/ml C3 for 1 hour at 37°C. The generated C3a was measured by the C3a EIA kit (Quidel). In some assays, patient’s or control IgG was added together with convertase components to analyze their effect on convertase formation. Convertase decay was determined after incubation of the formed convertase with the isolated IgG fractions alone or in the presence of 1 µg/ml FH for 1 hour at 37°C. Remaining convertase was detected with goat anti-FB antiserum.