AlphaLISA screening of RATP-Ig supernatants
RATP-Ig supernatants from glycan-base BG505-positive B cells were diluted into AlphaLISA buffer (PBS +0.05% Tween 20 + 0.5 mg/mL BSA), and 5 μL of each were transferred to an OptiPlate-384 assay plate (white opaque, PerkinElmer, Waltham, MA). To each well was added 10 μL of biotinylated Env trimer probe at 10 nM final concentration and 10 μL of anti-human Fc IgG (PerkinElmer, Waltham, MA) acceptor beads at 10 μg/mL final concentration. The mixtures were incubated at room temperature (RT) for an hour and then added 25 μL of streptavidin donor beads (PerkinElmer, Waltham, MA) at 40 μg/mL final concentration. The plate was incubated at RT for 30 min in the dark, and the AlphaLISA signal was read using a SpectraMax i3x multi-mode microplate reader (Molecular Devices, San Jose, CA). Only nine supernatants out of two plates of RATP-Ig supernatants were identified as double-positive for binding BG505 DS-SOSIP and glycan-base BG505, likely due to both false positives in B cell sorting and low level of Ig expression in RATP-Ig for some antibodies.