mAb-13G8 or mAb-QC03 (2.5 μg/ml) were diluted in 0.1 M carbonate buffer (pH 9.6), plated on a high-binding 96-well plate (Corning, Corning, NY) and incubated overnight at 4°C. Plates were blocked for 2 hours in blocking buffer [PBS with 0.05% Tween 20 (PBST) containing 3% milk/3% goat sera] at 37°C. Plates were washed four times in PBST and incubated with supernatant from transfected 293T cells at the indicated dilution in blocking buffer for 2 hours at 37°C. Plates were washed four times in PBST and incubated with an anti–M-segment antisera from DNA-vaccinated rabbits (diluted 1:1200) in blocking buffer and incubated at 37°C for 1 hour. Plates were washed four times in PBST and incubated with anti-rabbit IgG conjugated to horseradish peroxidase (1:1000; KPL) for 1 hour at 37°C. Plates were washed again four times in PBST, and 100 μl of SureBlue Reserve TMB microwell peroxidase 1-component (KPL) was added to each well. Reactions were stopped by adding 100 μl of TMB stop solution (KPL). The optical density (OD) at 450 nm was read on a Tecan microplate reader (Tecan Group Ltd.).

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