2.3 Receptor-binding analysis by HA assay
An α2,3-specific sialidase can effectively eliminate SAα - 2,3 Gal, but retain SAα - 2,6 Gal. With α2,3-sialidase-untreated TRBCs and α2,3-sialidase-treated TRBCs, the receptor-binding preference of the virus could be detected by HA’s change titer. HA assays using resialylated turkey red blood cells (TRBCs) were performed as described previously (Suptawiwat et al., 2008) with some minor modifications. Briefly, 1% TRBCs solution was divided into α 2,3-sialidase treated group and untreated group. α2,3-sialidase treated group was incubated with 1U 2,3-specific sialidase (Takara, Japan) at 37 °C for 12h, while the untreated group with phosphgate-buffered saline (PBS) as a mock control. The TRBCs were washed with PBS three times. After centrifugation, two groups of TRBCs were reconstituted to 0.6% concentration. Then receptor-binding preference of the virus was detected by HA assay. Complete elimination of the SAα-2,3-receptor on sialidase-treated TRBCs was confirmed by receptor staining and flow cytometry. The human influenza A virus A/ST/602/05(H3N2) and avian influenza virus DK/GX/767/2010 (H9N2) were used as controls for the HA assay.
2.4Receptor-binding analysis using a solid-phase direct-binding assay.
Viral receptor-binding specificity was determined using the solid-phase direct binding assay as described previously (Bi et al., 2020) with minor modifications. Briefly, 96-well microtiter plates were coated with biotinylated glycans α2-3-SA receptors (Neu5Acα2-3Galβ1-4GlcNAcβ-C3-PAA-biotin, 3’SLN) and α2-6-SA receptors (Neu5Acα2-6Galβ1-4GlcNAcβ-C3-PAA-biotin, 6’SLN) (GlycoNZ Corporation, MD, USA). Then the virus dilutions containing 64 HA units was added and the plates were incubated at 4℃ for 12 h. Virus-receptor binding reaction was detected with human antisera against influenza viruses HA and HRP-linked rabbit-anti-human antibody (Beyotime Biotechnology) The reaction was stopped with 100μl Stop Solution for TMB Substrate, and the absorbance was determined at 450 nm. The cutoff value for the glycan binding assays was the background value of the well with 100 ng of glycopolymer in the absence of added virus. The human influenza A virus A/ST/602/05(H3N2) and avian influenza virus DK/GX/767/2010 (H9N2) were used as controls for the solid-phase direct binding assay.