Blocking Assay for VLP-Glycan Interaction

To test the ability of each mAb to inhibit the interaction between the selected VLPs and glycans in vitro, we used a blocking assay. As previously described, we coated microtiter plates with 10 µg/mL of pig gastric mucin Type III (Sigma) for 4 hr at room temperature. Porcine gastric mucin (PGM) purified from porcine stomach mucosa contains both H and Lewis antigens, α-1,2-fucose and α-1,4-fucose^{24 (link),41 (link),42 (link)}. Plates then were blocked overnight at 4 °C in 5% nonfat dry milk. VLPs at 0.5 µg/mL were pretreated with serially diluted concentrations of each mAb for 1 h at room temperature. The optimal concentrations of mAbs were normalized before testing blocking ability and tested at concentrations beginning at 1000 nM and then diluted serially. VLP-mAb complexes were added to the PGM-coated and blocked microtiter plates. After 1 hr of incubation, the plates were washed three times with PBST and the same was done in between each step. Bound VLPs were tested using murine serum-containing anti-GI.3, GII.4, GII.6, or GII.17 polyclonal antibodies, followed by an HRP conjugated goat anti-mouse IgG human adsorbed antibody. Optical density was measured at 450 nm using a Synergy HT Microplate Reader (BioTek). Blocking studies also were repeated three times.