Antibody-Dependent Complement Deposition Assay

Antibody-dependent complement deposition (ADCD) was assessed as described previously [15 (link)]. In brief, biotinylated antigen was coupled to fluorescent NeutrAvidin beads (Thermo Fisher Scientific, Waltham, MA, USA). Plasma antibodies were diluted 1:10 in 0.1% BSA and incubated with the coupled antigen beads for 2 h at 37 °C. Beads were washed and incubated with complement factors from guinea pig for 20 min 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), and relative C3 deposition was analyzed by flow cytometry.