2.4 Solid-phase direct binding assay
Solid-phase glycan‐coated ELISA with the synthetic sialylglycopolymers
Neu5Acα2-3Galb1-4GlcNAcb1 (3’SLN)-PAA-biotin and Neu5Acα2-6Galb1-
4GlcNAcb1 (6’SLN)-PAA-biotin (GlycoTech) was performed to evaluate the
receptor binding properties of H7N9
AIV isolates as described previously (Li et al., 2014). An early poultry
isolate A/mallard/Huadong/S/2005
[HD/05(H5N1)] with specificity
for SAα‐2,3Gal, and a human isolate A/California/04/2009
[CA04(H1N1)] with specificity
for SAα‐2,6Gal, were used as controls, respectively (Li et al., 2014; H.
Sun et al., 2016).
2.5Thermal stability
In order to understand the thermal
stability of
H7N9
AIV isolates, virus-containing
allantoic fluids were
incubated at 56°C in a water bath
for various time intervals (0, 5, 10, 15, 30, 60, 90, 120, 150, 180 and
210 min) according to a previously described protocol (Imai et al.,
2012). Then, HA activity was analyzed by HA test against 1% chicken
erythrocytes in duplicate, while infectivity of viruses was assessed
by 50% tissue culture infective
dose (TCID50) on MDCK cells
(O’Donnell, Vogel, Matsuoka, Jin, &
Subbarao, 2014).