Neutralization Assay for Influenza Virus

Appropriately diluted IAVs (100 PFU) were incubated with serial dilutions of ch-rM2ss23 antibodies (0.01–100 µg/mL) and then inoculated into MDCK cells. Anti-HA MAb S139/1, which neutralizes both Adachi and Aichi [21 (link),32 (link)], was used as a positive control antibody. After incubation with the virus-MAb mixture, the cells were washed with PBS and overlaid with 0.3% BSA/MEM containing 1.2% Avicel RC 591 (FMC BioPolymer, Philadelphia, PA, USA) [33 (link)] and 5 µg/mL trypsin. After 20-h incubation, the cells were fixed with methanol and blocked with 1% BSA in PBS. Plaques were stained with MAb S139/1, HRP-conjugated goat anti-mouse IgG (H + L) (115-035-062, Jackson Immuno Research, West Grove, PA, USA), and a 3,3′-diaminobenzidine substrate (Wako, Osaka, Japan).