Antibody-Dependent Complement Deposition Assay

ADCD was determined by the deposition of the complement component C3b on the surface of CEM-NK_R.CCR5 cells as described in [10 (link)]. Target cells were pulsed with 10μg BG505.SOSIP.664 gp140 trimer in 100μl of R10 media (10% FBS 1% Pen/Strep RPMI, Gibco, Gaithersburg, MD) for 1 hour at room temperature and incubated with 100μg/ml of IgG preparation. HIV-negative plasma was used as a source of complement and diluted 1 in 10 in 0.1% gelatin/veronal buffer (Sigma-Aldrich, St Louis, MO) and 150μl added and incubated for 20 minutes at 37°C. The cells were then washed in 15mM EDTA in PBS and C3b was detected by flow cytometry using an anti-human/mouse complement component C3/C3b/iC3b mAb (Cedarlane, Burlington, Canada). Unpulsed cells were used as background controls and HIV-negative plasma was heat-inactivated at 56°C to remove complement as a negative control. The ADCD score was defined as geometric MFI multiplied by % cells positive for C3b deposition.