Antibody-Dependent Cellular Phagocytosis Assay

Antibody-dependent cellular phagocytosis (ADCP) was assessed in a bead-based assay (25 (link)). Antigens were biotinylated using Sulfo-NHS-LC-LC Biotin for 2h at room temperature (Thermo Scientific) and excess biotin was removed with a 3-kDa molecular mass cutoff column (Amicon/EMD, UFC500396). Yellow, fluorescent neutravidin beads (Thermo Fisher Scientific) were coupled to biotinylated ESAT-6/CFP-10, PPD and LAM for 2 h at 37°C. Subsequently, beads were washed and blocked with 1% BSA for 1h at room temperature. Then, antigen-coupled beads were incubated with 1:30 diluted plasma in PBS for 2 hours at 37°C. Following immune complex formation, samples were washed and 2.5x10⁴ THP-1 cells (ATCC) were added per well and incubated for 16 hours at 37°C in RPMI media with beta-mercaptoethanol. Following fixation, the next morning, sample acquisition was performed via flow cytometry (Intellicyt, iQue Screener plus) utilizing a robot arm (PAA), and analysis occurred using Forecyt software. Gating strategy included gating on THP-1 cells, single cells and FITC-positive events. A phagocytosis score was calculated as (percentage of bead-positive cells) x (MFI of bead-positive cells) divided by 10,000.