Saliva-based HuNoV Binding Assay

The saliva-based HBGA binding and blocking assays were performed as described previously [18 (link),26 (link),27 (link)]. To avoid potential HuNoV-specific antibodies in the saliva that may interfere with the receptor-binding assay, saliva samples were boiled before being used in the assays. The boiled human saliva samples with known HBGA phenotypes (A, B, or O) were diluted 1000-fold and coated on 96-well microtiter plates at 4 °C overnight. After blocking with 5% nonfat milk in PBS, HuNoV VLPs were added to a final concentration of 4 μg/mL. The bound VLPs were detected by using serially diluted IgYs (from 1:1000 to 1:128,000), followed by the addition of HRP-conjugated goat anti-chicken IgY at a dilution of 1:5000. The color was then developed by adding tetramethylbenzidine peroxidase liquid substrate (Kirkegaard and Perry Laboratory) and stopped after 10 min of incubation at 22 °C by adding 1 mol/L sulfuric acid. Optical density (OD) was measured at 450 nm with the use of an Epoch Micro-Volume Spectrophotometer System (BioTek, Winooski, VT, USA).