Virus-Sialylated Receptor Binding Assay

Purified virus binding to different sialylated receptor analogues was tested using an Octet RED biolayer interferometer (Pall ForteBio), as described previously (18 (link)). Receptor analogues contained 30-kDa polyacrylamide backbones conjugated to 20 mol% trisaccharides and 5 mol% biotin (Lectinity Holdings). The three analogues used in this study were α2,6-sialyllactosamine (6SLN), α-2,3-sialyllactosamine (3SLN), and Neu5Ac α-2,3Galβ1-4(6-HSO3)GlcNAc (3SLN[6su]). Sialoglycopolyemers were bound onto streptavidin-coated biosensors (Pall ForteBio) at ranges of concentrations from 0.01 to 0.55 μg/ml in HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20). Virus was diluted to a concentration of 100 pM in HBS-EP, 10 μM oseltamavir carboxylate (Roche), and 10 μM zanamivir (GSK). Virus association to the bound sialoglcopolymers was measured at 20°C for 30 min. Virus binding curves were normalized to fractional saturation and plotted as a function of sugar loading. Relative dissociation constants were calculated as described previously (18 (link), 25 (link)).