Antibody-Dependent Cell-Mediated Phagocytosis Assay

ADCP was measured using monocytic THP-1 cells (ATCC, Manassas, VA), as previously described (42 (link)). Briefly, CRF01_AE gp140 protein was biotinylated at a biotin to gp140 ratio of 50 following manufacturer’s instructions (Thermo Fisher, Waltham, MA) and excess biotin was removed using Zeba desalting columns (Thermo Fisher, Waltham, MA). Biotinylated gp140 was incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes, Eugene, OR) for 2 hours at 37°C. The gp140 beads were diluted 100-fold. In a 96-well plate, 10μl of diluted beads were mixed with diluted mother (1:3000) or infant (1:1000) plasma then incubated 2h at 37°C. Healthy donor plasma was utilized as a negative control. THP-1 cells (20,000 cells per well) were added and plates were incubated at 37°C for 18 hours. Cells were fixed with 4% paraformaldehyde solution and fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience, Franklin Lakes, NJ). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean fluorescence intensity of the bead-positive cells and dividing by 10⁴.