Purpose: To develop and evaluate a rapid plate-based HBGA-binding assay for human norovirus infectivity discrimination.
Methods: A sandwich-type HBGA capture assay using neutravidin plates, biotinylated synthetic HBGAs, and antibodies was optimized for detection of GII.4 Sydney Virus-Like Particles (VLPs). It was used for infectivity discrimination of VLPs exposed to heat (60°C to 80°C for one min) or copper (0-15 min). The results were compared to a traditional HBGA binding assay that was more complex and lengthy.
Results: The improved HBGA capture assay was capable of achieving a positive/negative (VLP/No VLP) ratio of 25.3±4.9 in < 2.5 hours with a detection limit of 0.1 µg/ml VLP. The assay performed in a manner similar to that of a much longer synthetic HBGA binding assay, with binding signal nearly completely abolished after heating VLPs at 75°C for one min (94.2±3.83% signal reduction). For all treatments, the loss of signal observed for the capture assay did not significantly (P>0.05) differ when compared to the more established assay. The new assay did not differ significantly (P>0.05) to the established assay for VLPs exposed to copper surfaces, with nearly complete loss (97.5±2.8%) of binding signal after 15 min.
Significance: This new HBGA capture assay allows for evaluation of norovirus receptor binding ability in under 2.5 hours. It is significantly faster, uses less reagents than previously reported assays, and is valuable for detection of infectious norovirus remaining after application of physical or chemical control methods.