ADCP Assay for Antibody-Dependent Cellular Phagocytosis

ADCP was measured as previously described [42 (link)]. Briefly, gp120 or gp70 V1V2 (Case A2) were biotinylated at a biotin (Thermo Scientific) to antigen ratio of 50:1 following manufacturer’s instruction and incubated with yellow-green strepavidin-fluorescent beads (Molecular Probes) 2h at 37°C. A 100-fold dilution of beads–gp120 (10μl) was incubated 2h at 37°C with 100μl of diluted plasma samples before addition of THP-1 cells (20,000 cells per well; Millipore Sigma, Burlington, MA, USA). After 19h incubation at 37°C, the cells were fixed with 2% formaldehyde solution (Tousimis, Rockvile MD USA) and fluorescence was evaluated on a BD LSRII instrument. The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean fluorescence intensity (MFI) of the bead-positive cells and dividing by 10⁴.