Peptide m coupled agarose beads

Human HIV-1 IgG antibodies used as reference controls are as follows: anti-V3^crown 10-188 (Mouquet et al., 2011 (link)); bNAbs 2G12 (Trkola et al., 1996 (link)), anti-CD4bs 3BNC117 (Scheid et al., 2011 (link)), anti-V3-glycan PGT121, PGT135, 10-1074, and BG8/BG18 (Freund et al., 2017 (link); Mouquet et al., 2012 (link); Walker et al., 2011 (link)); and anti-silent face SF12 (Schoofs et al., 2019 (link)). Non–HIV-1 antibodies include polyreactive and nonpolyreactive antibody ED38 (Meffre et al., 2004 (link)) and mGO53 (Wardemann et al., 2003 (link)), respectively. 10-1074 and mGO53 IgA monoclonal antibodies were also generated (Lorin et al., 2017 (link)). Recombinant IgG and IgA antibodies were produced by cotransfection of Freestyle 293-F cells (Thermo Fisher Scientific) using PEI precipitation method as previously described (Lorin and Mouquet, 2015 (link); Tiller et al., 2008 (link)) and purified by affinity chromatography using protein G Sepharose 4 fast flow beads (GE Healthcare) and peptide M-coupled agarose beads (InvivoGen), respectively. For competition ELISA experiments, purified antibodies were biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific).

All human sera were heat-inactivated at 56°C for 60 min. Human IgG and IgA antibodies were purified from donors’ sera by affinity chromatography using Protein G Sepharose 4 Fast Flow (GE Healthcare) and peptide M-coupled agarose beads (Invivogen), respectively. Purified serum antibodies were dialyzed against PBS using Slide-A-Lyzer Cassettes (10K MWCO; Thermo Fisher Scientific).