ADCD was determined by the deposition of the complement component C3b on the surface of CEM-NKR.CCR5 cells [3 (link)]. Target cells were pulsed with 6μg gp120 ConC, 14μg gp120 CAP45.G3 or 6μg gp140 C.ZA.1197MB in 100μl of R10 media (10% FBS 1% Pen/Strep RPMI, Gibco, Gaithersburg, MD) determined by titration as described above 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.
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