Anti-Pgp3 Antibody Detection Assay

Samples were diluted 1:400 in Buffer B (1X PBS [phosphate buffered saline], 0.5% casein, 0.5% polyvinyl alcohol [PVA], 0.8% polyvinylpyrrolidone [PVP], 0.3% Tween-20, 0.02% sodium azide and 3 µg/mL E. coli extract) and tested by MBA as described previously^{3 (link)}. Briefly, Pgp3-coupled MagPlex^® microspheres (Luminex, Austin, TX USA) were incubated with diluted sample for 1.5 h then washed with 0.05% Tween-20 in 1X PBS (PBST). Beads were washed with PBST and incubated with biotinylated mouse anti-human IgG and biotinylated mouse anti-human IgG4 for 45 min to detect anti-Pgp3 antibody bound to beads. After 3 additional washes with PBST, beads were incubated for 30 min with phycoerythrin-labeled streptavidin (SA-PE) to detect bound biotinylated anti-human IgG. After 3 more washes with PBST, loosely bound antibodies were removed with a final 30-min incubation in PBST containing 0.5% BSA and 0.02% sodium azide. Beads were washed one more time, resuspended in 1X PBS, and stored overnight at 4 °C. The next day, plates were read on a Bio-Plex 200 instrument (Bio-Rad, Hercules, CA, USA) equipped with Bio-Plex manager 6.0 software (Bio-Rad). The median fluorescence intensity (MFI) was recorded for each sample.