The ability of different FH fragments and mini-FH to destabilize the AP C3 convertase (decay acceleration activity) was evaluated using SPR, as described before (31 (link)). Briefly, C3b molecules were immobilized onto a CM5 sensor chip (GE Healthcare) by convertase-mediated deposition as described above. A mix of 100 nM FD and 500 nM FB in running buffer (HBS-P+) was injected for 3 min at 10 μl/min over the C3b surface to build the convertase complex (C3bBb) on the chip. Following an undisturbed decay of 1 min, the analytes (either FH, FH1-4, FH19-20 or mini-FH; all at 100 nM) were injected for 3.5 min. To regenerate the surface, residual convertases were decayed by consecutive injections of 2 μM FH1-4 and 1M NaCl. For comparative visualization of the pure decay acceleration response, SPR binding signals of the analytes in the absence of the convertase were subtracted from the corresponding convertase decay response. Processed, superimposed sensorgrams were normalized to compensate for the small drift in signal due to the convertase-mediated immobilization procedure in order to facilitate an overlay of sensorgrams at the time point of analyte injection (the normalization was below 7% for all sensorgrams). Duplicate sensorgrams are shown to demonstrate reproducibility.
To assess the cofactor activity of FH and constructs thereof, a fluid phase, time-course cofactor assay was performed in PBS similar to previous descriptions (31 (link)). Briefly, solutions containing FI and C3b (0.01 μM and 0.7 μM, respectively; Complement Technology) and either FH or mini-FH as cofactor (0.1 μM; added last) were prepared on ice and aliquoted into 20-μl aliquots prior to incubation at 37 °C for increasing amount of time (5, 10, 20, 40 min). A mixture in absence of any cofactors served as negative control. Each reaction sample was analyzed by 9 % SDS-PAGE, stained using Coomassie, and evaluated for the cleavage of the α’ chain of C3b (113 kDa) into the smaller fragments (43, 46 and 68 kDa) present in iC3b.
To assess the cofactor activity of FH and constructs thereof, a fluid phase, time-course cofactor assay was performed in PBS similar to previous descriptions (31 (link)). Briefly, solutions containing FI and C3b (0.01 μM and 0.7 μM, respectively; Complement Technology) and either FH or mini-FH as cofactor (0.1 μM; added last) were prepared on ice and aliquoted into 20-μl aliquots prior to incubation at 37 °C for increasing amount of time (5, 10, 20, 40 min). A mixture in absence of any cofactors served as negative control. Each reaction sample was analyzed by 9 % SDS-PAGE, stained using Coomassie, and evaluated for the cleavage of the α’ chain of C3b (113 kDa) into the smaller fragments (43, 46 and 68 kDa) present in iC3b.
