Biotinylated antiigg1b b682

ELISA plates were coated overnight at 4°C with 5 µg/ml of NP₁₆-bovine serum albumin (BSA), NP₄-BSA or CGG (Biosearch Technologies) in bicarbonate coating buffer (0.1 M sodium bicarbonate and 0.02% sodium azide at pH 9.6). Plates were washed with wash buffer (PBS containing 0.05% Tween 20) and after blocking 1hr with blocking buffer (PBS supplemented with 2% BSA and 0.05% Tween 20) at 37°C, serially diluted serum samples were added and incubated for 1 h at room temperature. Technical duplicates were performed for every serum sample. Plates were washed with PBS with 0.05% Tween 20 and incubated with 1 µg/ml biotinylated anti-IgG1^b (B68-2, BD Biosciences) for 1 hr followed by streptavidin conjugated horseradish peroxidase for 45 min. Peroxidase activity was detected by tetramethylbenzidine substrate (Dako) and the reaction was quenched with 2N H₂SO₄ and optical densities were quantified at 450nm. The end-point titer of each sample was determined using Prism software (GraphPad Software) from a one phase exponential decay curve defined as the dilution that generates an OD₄₅₀ value of the background plus 3 standard deviations.