EC50 binding analysis.

The EC₅₀ concentration for each antibody was determined as described previously (28). Briefly, we performed ELISA using plates coated with the HA of interest at 2 μg/ml overnight at 4°C and then blocked with 5% nonfat dry milk, 2% goat serum, and 0.1% Tween-20 in PBS for 1 hour. Three-fold dilutions of the mAb starting from 10 μg/ml were added to the wells and incubated for 1 hour, followed by incubation for 1 hour at 1:4,000 dilution of anti-human IgG alkaline phosphatase conjugate (Meridian Life Science, W99008A). The plates were washed 3 times between each step with PBS containing 0.1% Tween-20. Phosphatase substrate solution (1 mg/ml p-nitrophenol phosphate in 1 M Tris aminomethane) was added to the plates and incubated for 1 hour, and the optical density values were measured at 405-nm wavelength on a BioTek plate reader. Each dilution was done in triplicate, and the EC₅₀ values were calculated in Prism software (GraphPad) using nonlinear regression analysis.