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.