Phycoerythrin labeled neutralite avidin

A BAMA with C.1086 gp140 K160N-labeled magnetic beads (MagPlex; Bio-Rad) was used as previously described (72 (link)) to measure concentrations of antigen-specific IgG in secretions and IgA in both secretions and serum depleted of IgG. Briefly, beads reacted with dilutions of the standard (73 (link)) and specimens at 1,100 rpm at 4°C overnight were washed and developed with biotinylated anti-monkey IgG or -monkey IgA (Rockland) followed by phycoerythrin-labeled Neutralite-avidin (SouthernBiotech). The construction of standard curves and interpolation of antibody concentrations were done using Bioplex Manager software after measurement of fluorescence with a Bioplex 200 instrument (Bio-Rad). Concentrations of gp120-specific IgG or IgA in secretions were divided by the total IgG or IgA measured in the sample by an ELISA (74 (link)) to obtain the specific activity (nanograms of IgG or IgA antibody per microgram of total IgG or IgA).

A custom binding antibody multiplex assay (BAMA) assay with C.1086 gp140 K160N-conjugated Bio-Rad Bio-plex Pro magnetic COOH beads was used to quantify Env-specific IgG in rectal secretions and specific IgA in IgG-depleted sera and rectal secretions as previously described (Verma et al., 2019 (link)). Briefly, 50 µg gp140 was conjugated to 10x10⁶ beads as described (Iyer et al., 2015 (link)). The gp140-labeled beads (3500 per well) were shaken overnight at 1100 rpm and 4 °C with 10-fold dilutions of serum or secretion and standard Verma et al., 2019 (link). Beads were then washed with PBST and reacted for 30 minutes at room temperature with biotinylated goat anti-human IgG (Southern Biotech) or goat whole IgG containing anti-monkey IgA (Novus) followed by phycoerythrin-labeled neutralite avidin (Southern Biotech). Fluorescence was measured with a Bio-Rad Bioplex 200. Antibody concentrations were determined using standard curves prepared with Bioplex Manager software (Bio-Rad). Concentrations of gp120-specific IgG in secretions were divided by the concentration of total IgG or IgA measured in the sample by ELISA to obtain the specific activity (ng of gp140-specific IgG or IgA antibody per µg of total IgG or IgA, respectively).