Pgm type 3

To prepare a stock solution, 200 mg of type III PGM (Sigma-Aldrich, St Louis, MO, USA) was dissolved in 50 mL autoclaved distilled water, at room temperature (25 °C). The pH of the stock solution of PGM (4 mg/mL) was measured by using a digital pH meter (Thermo Scientific, Waltham, MA, USA). The stock solution was diluted with autoclaved distilled water, according to the required concentration of PGM for different assays.

VLPs bound to PGM type III (Sigma Aldrich, St. Louis, MO) or human type B saliva were detected by rabbit polyclonal antiserum (Cocalico Biologicals, Stevens, PA), as described previously.⁶⁶ (link)^,67 (link) For assays including bile salts, stocks were prepared in PBS, aliquoted, and stored at -20°C, and reagents were purchased from Sigma Aldrich: bovine bile (B3883), GCDCA (G-0759), and TCA (T4009). Data were graphed by single-site binding curve analysis in GraphPad Prism 8.1.1 (San Diego, CA).⁶⁸

As described previously [20 (link),25 (link)], EIA plates were coated with 10 μg/ml PGM type III (Sigma Aldrich) diluted in PBS and blocked with 5% Blotto in PBS/0.05% Tween 20. VLPs (0.5 μg/ml) were pretreated with decreasing concentrations of serum for 1 h before being added to the carbohydrate-ligand-coated plates for 1 h. Ligand-bound VLP was detected with rabbit anti-GI or -GII or -GII.4 VLP hyperimmune serum followed by anti-rabbit-IgG-HRP (GE Healthcare). All tested VLPs, except for GII.4.2012 and GII.4.2006b.P.D302, were components of the VLP cocktails used to immunize rabbits. Multivalent GII.4 VLP immunization resulted in broadly reactive serum that recognizes GII.4.2012 and GII.4.2006b.P.D302 [44 (link)]. Wash steps, Ab dilutions, and color development were completed as described above. All incubations were done at room temperature. The percent control binding was defined as the binding level in the presence of Ab pretreatment divided by the binding level in the absence of Ab pretreatment multiplied by 100. Blockade data were fit using sigmoidal dose–response analysis of nonlinear data in GraphPad Prism 6.02. EC₅₀ values were calculated for sera that demonstrated blockade of at least 50% at the dilution series (40–20480) tested. Sera that did not block 50% of binding at the highest concentration tested were assigned an EC₅₀ of 20 for statistical comparison.

A surrogate blocking assay was used to measure neutralizing antibodies. Ninety-six-well plates were coated with 5 μg/mL pig gastric mucin (PGM) Type III (Sigma‒Aldrich) in PBS, incubated for 4 h at room temperature, and then blocked with 5% no-fat milk in PBST at 4 °C overnight. Serially diluted antiserum samples were mixed with an equal volume of 2 µg/mL VLPs and incubated for 1 h at 37 °C. One serum-free well was used as a control for calculating the blocking rate. Then, the mixtures were added to the PGM-coated plates and incubated for 1 h at 37 °C. After washing three times, the plates were incubated with rabbit anti-VLP antibody (Absolute antibody, UK) for 1 h at 37 °C, followed by incubation with HRP-conjugated anti-rabbit IgG for 1 h at room temperature. After color development, the absorbance was determined at 450 nm. The blocking rate was calculated as (mean OD₄₅₀ treated without serum − mean OD₄₅₀ treated with serum)/mean OD₄₅₀ treated without serum × 100%.

BLG from bovine milk, BSM (Type I-S), and PGM (Type III) were purchased from Sigma Aldrich (Brøndby, Denmark), and were used as received. Protein solutions were prepared by dissolving proteins in 100 mM PBS solutions. By addition of HCl as appropriate, the pH values of the buffer solutions were adjusted to pH 7, 5, and 3 where the BLG is negatively, neutral, and positively charged, respectively [4 (link)]. All buffer solutions were filtered (polyethersulfone 0.20 mm).

The presence of serum IgG antibodies that block binding of NoV VLPs to the HBGA carbohydrates were determined in ELISA-based blocking assay according to previously published protocols [23 (link)]. Group-wise pooled mouse sera were examined for capability to block VLP binding on HBGAs present in pig gastric mucin (PGM, type III, Sigma-Aldrich, Saint Louis, MO, USA, Cat. M1778) [23 (link),34 (link)]. Serum two-fold dilutions were pre-incubated with 0.1 µg/mL GI.4 or GII.4-2006a NoV VLPs in sample buffer (1% milk in PBS + 0.05% tween) prior to plating on PGM-coated (2 µg/mL) and blocked (5% milk in PBS) microwell plates (Corning Inc., Corning, NY, USA, Cat. 3690). Following 1 h incubation at +37 °C, bound VLPs were detected with rabbit polyclonal anti-NoV antisera (ICON Genetics, Halle, Germany) followed by anti-rabbit IgG-HRP antibody (Abcam, Cambridge, UK, Cat. ab97051) and OPD substrate. Maximum HBGA binding of VLPs was determined in wells with VLPs lacking the serum. The blocking index (%) was calculated as follows: 100% − [(OD490 of wells with VLP and serum/OD490 of maximum binding wells) × 100%]. A 50% blocking titer (BT₅₀) was determined as the reciprocal of the highest serum dilution able to block at least 50% of VLP-HBGA binding.