2.3. Influenza Hemagglutination Inhibition (HAI) Assay

Hemagglutination inhibition (HAI) assays were performed using the influenza A/H3N2 virus (A/Singapore/INFIMH-16-0019/2016/H3N2 strain), in accordance with WHO guidelines [20] and following an optimized protocol, as previously published [17]. Briefly, sera were treated with receptor-destroying enzyme (RDE) from Vibrio cholerae filtrate (Sigma-Aldrich, St. Louis, MO, USA) to eliminate non-specific inhibitors of hemagglutination. Treated sera were two-fold serially diluted with PBS in a V-bottom 96-well plate. Diluted sera (25 µL) were mixed with 25 µL of standardized influenza A/H3N2 virus solution containing 4 hemagglutination units (HAU) and incubated for 30 min at room temperature. Then, 0.5% turkey red blood cell suspension (50 µL) was added to the mixture. The HAI titer were determined after 45 min incubation and defined as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. All dilutions were tested in triplicate. A positive pooled control serum was run with each batch of samples. The coefficient of variation (CV) of the HAI assay in our laboratory was 2.9% [21].