Ap conjugated goat anti mouse igg antibody

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.

Binding of FHR1 with surface-bound C3b was detected as previously reported (24 (link)). In brief, Saccharomyces cerevisiae strain (Mingzhoubio B47202) cells were resuspended in TBS and added in the microtiter plates 200μl per well for about 45min RT for deposition. After washing away the extra cells with 0.02%TBST and blocking with 3% milk in 0.02%TBST for 2 hours at 25°C, 100nM C3b or TBS were added and incubated for 1 hour at 37°C. And after incubation with serial dilution of FHR1*A and FHR1*B (5μg/ml-1.25μg/ml), mouse anti-human FHR1 antibody (R&D, cross-reacts with FH), and AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich) were added for incubation in succession. In the last, alkaline phosphatase chromogenic substrate were added and the optical density was read at 405 nm.