Quantifying Antibody-Mediated Complement Activation

As described recently (32 (link)), biotinylated antigen gp120 MN was coupled to red fluorescent neutravidin beads (Thermo Fisher Scientific). Immune complexes were formed with plasma samples for 30 minutes at 37°C at 3 dilutions (1:100, 1:500, 1:2500) in order to calculate the AUC. Lyophilized guinea pig complement was resuspended according to manufacturer’s instructions (Cedarlane), and 2 μL per well was added in veronal buffer with 0.1% gelatin (Boston BioProducts). After 20 minutes of incubation at 37°C, immune complexes were washed and C3 was detected with a fluorescein-conjugated goat IgG fraction to guinea pig complement C3 (catalog 855385, MpBio). Complement-coated beads were acquired on a BD LSR II with a high-throughput sampler, and C3 deposition was reported by gating on single beads and C3⁺ events of 2 independent runs. AUC was calculated in Prism.