Enzyme-linked lectin assay.

An enzyme-linked lectin assay (ELLA) was used to assess neuraminidase-inhibiting antibodies as previously described (74,–76) with minor variations. As recombinant H9N2 viruses were used to generate antisera specific for swine N2 of interest, they could not be used as antigens in the ELLA, and thus, we used wild-type swine or human IAV (H1N2 or H3N2) as antigens for ELLA experiments. Briefly, 2-fold serial dilutions of H9N2 antisera were incubated with wild-type H1N2 or H3N2 IAV on fetuin-coated plates for 18 to 20 h at 37°C. Following incubation, plates were washed with PBS-Tween, incubated with horseradish peroxidase (HRP)-conjugated peanut agglutinin (Sigma-Aldrich, St. Louis, MO), and detected with TMB substrate (KPL Laboratories, Gaithersburg, MD). Plates were read at 650 nm and optical density at 650 nm (OD₆₅₀) values were used to calculate the percent inhibition. The inverse of the highest serum dilution that showed a 50% inhibition of NA activity was considered the NA inhibition (NI) titer.