The ADCP assay was adapted from (39 (link)). Briefly, gp140 SHIVSF162p3 and the recombinant Mymetics gp41 antigens were biotinylated using sulfo-NHS LC-LC biotin, coupled to yellow-green, fluorescent Neutravidin 1 μm beads (Invitrogen, F8776) for 2 h at 37°C and washed two times in 0.1% bovine serum albumin (BSA) in PBS. Ten μl/well of coupled beads were added to 96-well plates with 10 μl/well of diluted sample for 2 h at 37°C to form immune complexes. After incubation, the immune complexes were spun down, and supernatants were removed. THP-1 cells were added at a concentration of 2.5 x 104 cells/well and incubated for 18 h at 37°C. After incubation, the plates were spun down, the supernatant was removed, and cells were fixed with 4% paraformaldehyde (PFA) for 10 min. Fluorescence was acquired with a Stratedigm 1300EXi cytometer. Phagocytic score was calculated using the following formula: (percentage of FITC+ cells) * (the geometric mean fluorescent intensity (gMFI) of the FITC+ cells)/10,000. A polyclonal HIVIg pool available from the NIH AIDS Reagent Program was used as a positive control. Serum from a human HIV-seronegative donor was used as negative control.
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