Tetramethylbenzidine

ELISA plates were coated with 5 μg/ml NP₁₆ or NP₄ or with WNV envelope (E) protein overnight at 4°C in coating buffer containing 0.1 M sodium bicarbonate and 0.02% sodium azide at pH 9.6. Plates were washed three times with ELISA wash buffer (0.05% Tween 20 in PBS). Plates were blocked for 1 hour at room temperature with 2% BSA/PBS blocking buffer. Sera was then serially diluted in blocking buffer, plated and incubated for 1 hour at room temperature. Plates were washed three times followed by incubation with 1μg/ml biotinylated anti-mouse IgG (Jackson Immunoresearch), anti-IgG1^a-, or anti-IgG1^b (BD Biosciences) for 1 hour at room temperature. Wells were washed three times and then streptavidin-conjugated horseradish peroxidase (BD Biosciences) was added to each well and incubated for 1 hour at room temperature. Wells were washed three times with wash buffer and one time with PBS. Peroxidase activity was detected using tetramethylbenzidine (Dako) and quenched with 2 N H₂SO₄. Optical densities were measured at 450 nm. The end-point titer of each sample was calculated by a one-phase exponential decay curve and defined as the dilution that generated an OD₄₅₀ value that was 3 standard deviations above background. Prism software (GraphPad Software) was used to calculate the end-point titer.