Pig Gastric Mucin Type III (PGM) (Sigma Chemicals) has been validated as a substrate for NoV VLP antibody-blockade assays [17] . PGM contains relatively high levels of H and A antigen and more moderate levels of Lewis Y antigen [17] . All of the GII.4 VLPs used in the blockade assays in this study bind to both PGM and synthetic Bi-HBGA, and binding to PGM is consistent with synthetic Bi-HBGA binding profiles for a-1,2-fucose (H antigen) and a-1,4-fucose (Lewis antigen) containing molecules [13, 17, 77] . For blockade assays, PGM was solvated in PBS at 5 mg/ml and coated onto EIA plates at 10 mg/ml in PBS for 4 hours and blocked over night at 4uC in 5% dry milk in PBS-0.05% Tween-20. VLPs (0.5 mg/ml) were pretreated with decreasing concentrations of test mAb or donor plasma for 1 hour at room temperature before being added to the carbohydrate ligand-coated plates for 1 hour. Bound VLP was detected by a rabbit anti-GII norovirus polyclonal sera made from hyperimmunization with either GII.4.2009 or a cocktail of GII. 4.1997 , GII.3.1999 , GII.1.1976 , and GII.2.1976 VLPs, followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). The percent control binding was defined as the binding level in the presence of antibody pretreatment compared to the binding level in the absence of antibody pretreatment multiplied by 100. All incubations were done at room temperature. Each step was followed by washing with PBS-0.05% Tween 20 and all reagents were diluted in 5% dry milk in PBS-0.05% Tween-20. All antibodies were tested for blockade potential against the panel of GII.4 VLPs at two-fold serial dilutions ranging from 0.08 to 2 mg/ ml. Additional concentrations of blockade antibodies were tested if needed to complete the sigmoid dose-response curve. Blockade of synthetic Bi-HBGAs (Glycotech, Gaithersburg, MD) assays were done as described for PGM with the following exception. Bi-HBGAs were bound to Neutri-avidin coated plates (Pierce) at 10 mg/ml for one hour prior to the addition of 1 mg/ml VLP for 1.5 h. Reported mean % control binding reflects the results of at least two independent experiments with each dilution tested at least in duplicate. An antibody was designated as a ''blockade'' antibody for a VLP if at least 50% of control binding was inhibited by 2 mg/ ml antibody. Blockade data were fitted and EC50 values calculated using Sigmoidal dose response analysis of non-linear data in GraphPad Prism 5 (www.graphpad.com). EC50 values between VLPs were compared using the One-way ANOVA with Dunnett post test, when at least three values were compared or the unpaired t-test when two values were compared. A difference was considered significant if the P value was ,0.05. To test for antibody binding that prevents detector antibody from binding to the VLP instead of the VLP binding to the PGM, select blockade assays are performed without using a detector antibody and instead developed directly with an anti-human IgG-HRP. Antibodies tested this way give two responses; 1) a bell-shaped response curve for antibodies that are blockade and 2) a sigmoidal shaped curve for antibodies that are not blockade. These data indicate that it is the amount of human mAb that is directly blocking the VLP from binding to PGM. Of note, VLP concentrations in blockade assays are in the low nanomolar range and therefore cannot discriminate between antibodies with sub-nanomolar affinities.