Alkaline phosphatase chromogenic substrate

To assess the capacities of FHR1*A and FHR1*B to bind C3b, we conducted solid-phase C3b binding assays. Serial dilutions of the recombinant FHR1*A and FHR1*B proteins (2.5 μg/ml-0.078 μg/ml in PBS) were coated onto plates (Thermo Fisher Scientific) and incubated at 4°C for more than 16 hours. After washing with 0.1% PBST and blocking with 1%BSA/PBST at 37°C for 1 hour, C3b (2 μg/ml) was added and incubated at 37°C for 1 hour. Then, FHR1-bound C3b was detected using a C3c polyclonal antibody (Dako), followed by the addition of an anti-rabbit IgG-alkaline phosphatase antibody (Santa Cruz). In the reverse setting, 100nM C3b (Complement Tech) dissolved in TBS (140mM NaCl, 2mM CaCl₂, 1mM MgCl₂ and 10mM Tris, pH 7.4) were immobilized in the microtiter plates at 4°C overnight. After blocking with 3% milk in 0.02%TBST for 2 hours at 25°C, serially diluted FHR1-A and FHR1-B proteins (10μg/ml-1.25μg/ml) were added and incubated for 30min at 37°C. And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich). Finally, the plates were developed with alkaline phosphatase chromogenic substrate (Sigma-Aldrich), and the optical density was read at 405 nm.

To compare the capacity of FHR1*A and FHR1*B to compete with FH to bind C3b, FH (9 μg/ml) was immobilized on the surface of a microtiter plate (Thermo Fisher Scientific) at 4°C overnight. After blocking with 1% BSA/PBST at 37°C for 1 hour, serial dilutions of the recombinant FHR1*A or FHR1*B proteins (2.5 μg/ml-0.039 μg/ml) as well as C3b (0.25 μg/ml) were added to the plates. After shaking for 30 sec, the plates were incubated at 37°C for 1 hour. After washing with 0.1%PBST, FH-bound C3b was detected with a C3c polyclonal antibody (Dako), followed by incubation with an alkaline phosphatase-conjugated anti-rabbit IgG antibody as the secondary antibody (Sigma-Aldrich). The plates were then developed with alkaline phosphatase chromogenic substrate (Sigma-Aldrich), and the optical density was read at 405 nm.