Enzyme-linked lectin assay

Enzyme-linked lectin assay (ELLA) was used to quantify neutralization of neuraminidase (NA) enzymatic activity by Ab as described before (21). Briefly, NUNC Immuno 96-microwell plates (Sigma-Aldrich) were coated overnight at 4°C with 25 μg/ml fetuin (Sigma-Aldrich) in PBS containing 0.02% sodium azide. Heat-inactivated sera and BAL were serially diluted in DMEM supplemented with 0.1% BSA, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM l-glutamine starting at 1:40 and 1:4, respectively. H7N1 S-FLU [eGFP/N1(A/Eng/09)] H7(Netherlands/219/2003) was used to minimize any potential steric effect of Abs binding to H1 HA. An optimal concentration of H7N1 S-FLU was added to the diluted Abs for 20 min on a plate shaker. One hundred microliters of the mixture of virus and diluted samples were then transferred to the washed coated plate and incubated for 18 h at 37°C. Peanut agglutinin–conjugated with HRP (Sigma-Aldrich) was added at 1 μg/ml in PBS and incubated at room temperature for 2 h. The plates were washed and developed with 50 μl of TMB (BioLegend); after 5 min, the reaction was stopped with 50 μl of 1 M sulfuric acid and absorbance measured at 450 and 630 nm. The 50% inhibition titer was calculated as the highest dilution above the IC₅₀ line (midpoint between the signal generated by virus-only and medium-only wells).