Mucin Binding Assay for Influenza Hemagglutinin

Mucin binding assay was performed as described by Nakamura et al. with some modifications [22] (link). The wells of 96-well microtiter plates were coated with 10 µg of bovine submaxillary mucin (BSM) or 1 µg of porcine gastric mucin (PGM) (Sigma Aldrich). After washing with PBS containing 0.2% Tween20 and blocking with 1% BSA, mucin-coated wells were incubated with 1 µM GST-tagged HA1, HA3, or 50 nM HA complex. For the removal of sialic acid, the wells were pretreated with 5 mU/ml Arthrobacter ureafaciens neuraminidase (Nacalai Tesque) for 1 h at 37°C before the addition of HA. Bound proteins were probed with rabbit anti-GST antibody (Sigma Aldrich) or rabbit anti-B16S antibody [15] (link), followed by peroxidase-labeled goat anti-rabbit IgG (Jackson Immunoresearch). The plates were developed using ABTS (Roche Diagnostics), and absorbance was measured at 405 nm using a microplate photometer (Thermo Scientific).