Quantifying Antibody-Mediated Complement Activation

Antibody-dependent complement deposition (ADCD) was assessed as described previously [41 (link)]. Briefly, biotinylated BG505 protein was coupled to fluorescence NeutrAvidin beads (Thermo Fisher Scientific). Mouse sera antibodies were diluted 1:10 in 0.1% BSA and incubated with the coupled BG505 beads for 2 hrs at 37°C. Beads were washed and incubated with complement factors from guinea pig (Sigma-Aldrich) for 20 mins at 37°C. The complement reaction was then stopped by washing with 15 mM EDTA in PBS. C3 deposition on the beads was detected with a 1:100 diluted FITC-conjugated anti-guinea pig C3 polyclonal antibody (MP Biomedicals, Cat: 0855385) and relative C3 deposition was analyzed by flow cytometry.