Pig gastric mucin

Pig gastric mucin (Sigma-Aldrich) was immobilized at 10 μg/ml in PBS on 96-well-plate at 4°C overnight. Wells were washed with PBS-0.05% Tween 20 (PBS-T) three times. After blocking with 5% non-fat milk at RT for 1 hour, plates were washed once with PBS-T. Fc-huCD300 proteins (10 μg/ml) were mixed with purified HuNoV VLPs at indicated concentrations and then added and incubated for 1 hour at RT. The wells were washed three times with PBS-T followed by addition of rabbit anti-VP1 sera diluted in PBS (1:2000). Wells were incubated at RT for 1 hour, followed by washing with PBS-T three times as described previously[44 (link)]. Secondary goat anti-rabbit-HRP diluted in PBS were added and incubated for 1 hour. After five washes, TMB substrate was added, and 2M H₂SO₄ was applied to stop the reaction. Absorbance at 450 nm was measured.

Pig gastric mucin and fluorescein isothiocyanate-labeled 4.4-kDa dextran (FD4) were both purchased from Sigma-Aldrich (St. Louis, MO, United States). Kits for plasma total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), and a kit for catalase (CAT) activity were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). ELISA kits for tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), lipopolysaccharide (LPS), and lipopolysaccharide-binding protein (LBP) were purchased from Huijia Biotechnology Co., Ltd. (Xiamen, China). The BCIP/NBT kit for intestinal alkaline phosphatase (IAP) was purchased from Beyotime Biotechnology (Jiangsu, China). The other solvents and reagents were all analytical grade (Sinopharm Chemical Reagent, Shanghai, China).

A dynamic retention method previously developed by Khutoryanskiy and coworkers (Cave et al., 2012 (link), Cook et al., 2015 (link), Irmukhametova et al., 2011 (link), Withers et al., 2013 (link)) was adapted for this experiment. The retention experiment allows indirect quantification of the amount of sample retained on a mucosal surface after being repeatedly washed with an artificial eluent. To visualise retention of the sample sodium fluorescein (0.01%) was added to the solutions prior to placement on the tissue. For this experiment, ex vivo porcine tongue was used as the mucosal surface and an artificial saliva (AS) formulation was used as adapted from Madsen, Sander, Baldursdottir, Pedersen, and Jacobsen (2013) (link), as the eluent. This AS recipe was found to best simulate the retention profile achieved with real human saliva (Madsen et al., 2013 (link)). The AS was comprised of CaCl₂ (4 mM), KCl (10 mM), NaHCO₃ (2 mM), NaCl (7 mM), KH₂PO₄ (6.7 mM) and pig gastric mucin (2.5 % w/v) (Sigma Aldrich Poole, UK).