Snap system

Total purified IgG (100 μg/ml) were incubated for 45 min at 37°C with C3 (25 μg/ml), FB (0 to 50 ng), FD (0.05 μg/ml) (all from Complement Technologies, Tylor, Texas) in Hepes, 40 mM NaCl supplemented with 10 mM MgCl₂. The reaction was stopped by adding DTT-containing sample buffer. The cleavage of C3 was probed by a Western blot, using SNAP system (Millipore). After blocking with Tris 10 mM, NaCl 150 mM, 0.1% Tween, 1% BSA, blots were probed with a 1:5,000 dilution of goat anti-human C3 IgG (Calbiochem) followed by HRP-conjugated rabbit anti-goat IgG (Santa Cruz). The signal was developed by chemiluminescence using ECL kit (Perkin Elmer) and MyECL Imager (Thermo Scientific). Percentage of C3 cleavage revealing convertase formation was characterized by the appearance of α'-band and quantitated by densitometry of the scanned images. The ratio between α' and the β bands was calculated at 50 ng of FB. The same experiment was reproduced with monoclonal and polyclonal Ig fractions of 5 patients and with IgG of 3 MIg-C3GN patients after chemotherapy adapted to the B cell clone. IgG from 8 patients with MIg without kidney disease were used as controls. The same experiment was reproduced with monoclonal and polyclonal fractions of 5 MIg-C3G patients IgG.

C3 protein (20 μg/ml; Calbiochem) was incubated at 37°C for 0, 1, 5, or 10 min with FI (10 μg/ml; Complement Technologies) and FH (20 μg/ml; Complement Technologies, Tylor, Texas), or soluble CR1 (10 μg/ml; RandD Systems) in 10 mM Tris, 150 mM NaCl, pH 7.4 in presence of 100 μg/ml of total purified IgG. Samples were boiled and the cleavage of the C3 was probed by a Western blot, using SNAP system (Millipore). After blocking with Tris 10 mM, NaCl 150 mM, 0.1% Tween, 1% BSA, the blots were probed with a 1:5,000 dilution of goat anti-human C3 IgG (Calbiochem) followed by HRP-conjugated rabbit anti-goat IgG (Santa Cruz). The signal was developed by chemiluminescence using ECL kit (Perkin Elmer) and MyECL Imager (Thermo Scientific). Cleavage efficiency was evaluated by the appearance of the α43 band and the disappearance of the α-chain at 10 min and quantitated by densitometry of the scanned images. The ratio between α43 and the β bands (representing the % of C3b cleaved) was plotted vs. the time of incubation.