Quantifying Complement Deposition Assay

Complement deposition was measured as described previously with a bead-based assay [51 (link)]. Briefly, biotinylated antigen gp140 ConS was coupled to red fluorescent neutravidin beads (Thermo fisher). Plasma samples were diluted 1:10 in PBS and allowed to form immune complexes with antigen coupled beads for 2 h at 37°C. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane) and 4 μl per well was added in gelatin veronal buffer containing Mg²⁺ and Ca²⁺ (GVB⁺⁺, Boston BioProducts) and incubated with the washed immune complexes for 20 min at 37°C. C3 deposition was detected with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (MpBio). Complement coated beads were acquired via flow cytometry (IntelliCyt, iQue Screener plus) and C3 deposition was reported by gating on single beads and C3 positive events of two independent runs.