Trypsin Susceptibility Assay for Influenza HA

For the trypsin susceptibility assay, 0.5 μM HA was pre-incubated at pH 8.0 with 2.5 μM iHA-100, 1.0 μM 14A7, or 1.0 μM CR6261 for 30 min at room temperature^{18 (link)}. 14A7 is an HA head binding antibody produced in-house^{29 (link)}. CR6261 is an HA stalk binding antibody that protects HA from pH-induced proteases^{17 (link)}. Control reactions contained 2% DMSO without iHA-100. The pH of each reaction was lowered to pH 4.9 using 1 M sodium acetate buffer, while one reaction was retained at pH 8.0 to examine trypsin digestion at near-neutral pH. The reaction solutions were then thoroughly mixed by pipetting and incubated for 30 min at 37 °C. After incubation, the reaction solutions were neutralized to pH 8.0 with 1 M Tris-HCl buffer. Trypsin-ultra (New England Biolabs, Ipswich, MA) was added at a final ratio of 1:50 by mass, and the samples were digested for 20 min at 37 °C. Trypsin digestion was quenched by the addition of SDS loading buffer, and samples were boiled for 3 min at 99 °C. Finally, all samples were analyzed with 4–20% SDS-PAGE and immunoblotted with rabbit anti-H5HA antibody (Pep-10#11, produced in-house).