NoV GII.4 Antibody Blocking Assay

In order to determine the surrogate neutralization ability of NoV GII.4-specific antibodies, a blocking assay was conducted according to a published protocol [24 (link)] using human saliva type A as the source of HBGAs [42 (link)]. Individual mouse sera (diluted 1 : 50) or groupwise pooled sera (titrated from 1 : 50) were preincubated with 0.1 μg/ml GII.4 VLPs for 1 h at +37°C in low-binding tubes (Eppendorf, Hamburg, Germany) prior to adding the preparations on saliva-coated plates. The bound VLPs were detected using human NoV-positive serum (1 : 4000) and horseradish peroxidase- (HPR-) conjugated anti-human IgG antibody (1 : 6000, Novex, Invitrogen) reacting with OPD substrate (Sigma-Aldrich). The OD readings were measured as described above. Wells incubated with VLPs lacking mouse sera were added to each assay to determine the maximum binding OD. The blocking index (%) was calculated as 100% − [(OD wells with VLP‐serum mix/maximum binding OD) × 100%]. The results are expressed as the mean blocking indexes of individual mice for each immunization group or as mean blocking indexes of replicate wells if groupwise pooled sera were used.