Pig gastric mucin type 3

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.

EIA and blockade antibody assays were performed at described (Lindesmith et al., 2018a (link)) at 0.25 μg/ml VLP. EIA plates were coated with VLP in PBS for 4 h and blocked over night at 4°C in 5% dry milk in PBS-0.05% Tween-20 before the addition of decreasing two-fold serial dilutions of mAb at 37°C. Bound mAb was detected by anti-human IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). Similarly, VLPs were pretreated with decreasing concentrations of mAb for 1 h and added to pig gastric mucin type III (Sigma Aldrich, St. Louis, MO) coated plates for 1 h. Bound VLP was detected by anti-VLP rabbit hyperimmune sera followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed as above. Percent control binding is defined as the binding in the presence of antibody pretreatment compared to the binding in the absence multiplied by 100.

VLPs (0.25 μg/ml) were pretreated with decreasing concentrations of MAb for 1 h and added to wells coated with pig gastric mucin type III (Sigma-Aldrich, St. Louis, MO) for 1 h. Bound VLP was detected as described above using anti-VLP rabbit hyperimmune sera. Percent control binding is defined as the level of binding in the presence of antibody pretreatment compared to the level of binding in the absence of antibody multiplied by 100. The blockade data were fit using sigmoidal dose-response analysis of nonlinear data in GraphPad Prism 702. EC₅₀ and Hill slope values were calculated for antibodies that demonstrated blockade of at least 50% at the dilution series tested. Antibodies that did not block 50% of binding at the highest dilution tested were assigned an EC₅₀ of two times the assay upper limit of detection for statistical comparison (9 (link)). Increasing antibody-VLP-ligand incubations to 40°C (simulating fever) decreases the blockade EC₅₀ about twofold, or one dilution, in preliminary studies.