Yellow green streptavidin fluorescent beads

ADCP was measured as previously described (65 (link)). Briefly, gp120 CM235 (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 CM235 gp120 Recombinant Protein, ARP-12816, contributed by NIAID, DAIDS) was biotinylated at a biotin-to-protein ratio of 50 according to the manufacturer’s instructions (Thermo Fisher Scientific) and incubated with yellow-green streptavidin fluorescent beads (Molecular Probes) for 2 hours at 37°C. Ten microliters of a 100-fold dilution of beads-protein was incubated for 2 hours at 37°C with 100 μL of 200-fold diluted plasma samples before addition of Tamm-Horsfall protein 1 (THP-1) cells (20,000/well; MilliporeSigma). After an 18-hour incubation at 37^oC, the cells were fixed with 4% formaldehyde solution (Tousimis), and fluorescence was evaluated on an LSR II (BD Biosciences). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geo MFI of the bead-positive cells and dividing by 10⁴.

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.

ADCP was measured as previously described (37 (link)). Briefly, biotinylated SARS-CoV-2 spike proteins from D614G and BA.1 (both from Sino Biological) were incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes) for 2 hours at 37°C. Ten microliters of a 100-fold dilution of beads–protein was incubated for 2 hours at 37°C with 100 μl of a 1000-fold diluted plasma samples before the addition of THP-1 cells (25,000 cells per well; MilliporeSigma, Burlington, MA, USA). After 19 h of incubation at 37°C, the cells were fixed with 2% formaldehyde solution (Tousimis, Rockville, MD, USA), and fluorescence was evaluated on an LSR II (BD Biosciences). 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 the product by 10⁴.