A competition ELISA assay was conducted to determine the level of F10-like Ab in the serum samples. F10 Ab (human IgG1) was biotinylated with Sulfo-NHS-SS-Biotin (Pierce Biotechnology) in accordance with the manufacturer's instructions. Biotinylated F10 (Bio-F10; 3 ng/mL) was mixed at 1:1 (vol) ratio with serum samples at various dilutions and added to ELISA plates coated with H5-VN04 trimer. The competition of serum samples for the binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin (BD Bioscience).