Influenza HA Receptor Binding Assay

We tested the receptor binding specificity of the HA protein for α2,3- or α2,6-linked sialic acid using a solid-phase binding assay with two different glycopolymers: α-2,6 glycans (6′SLN, Neu5Aca2-6Galb1-4GlcNAcb-PAA-biotin) and α-2,3 glycans (3′SLN, Neu5Aca2-3Galb1-4GlcNAcb-PAA-biotin), as previously described (Imai et al., 2012 (link)). Briefly, a streptavidin-coated high-binding capacity 96-well plate (Pierce) was incubated with PBS containing different concentrations (starting from 2.4 μM) of biotinylated glycans at 4°C overnight. After the glycan solution was removed, the plates were washed four times with ice-cold PBS and then incubated at 4°C overnight with PBS containing 128 HA units of purified influenza virus. After washing, the plates were incubated for 4 h at 4°C with mouse antibody against influenza NP. The plates were then washed four times and incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (Sigma-Aldrich) for 2 h at 4°C. After four washes, the plates were finally incubated with tetramethylbenzidine substrate (Thermo Scientific), and the reaction was stopped with 50 μl of 2 M H2SO4. Absorbance was determined at 450 nm.