Carbohydrate-Ligand-Binding Assay for VLPs

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.