Anti mouse immunoglobulin igg1a

IL-2, IL-4, IL-5, and IL-13 were analyzed by enzyme-linked immunosorbent assay as described by the manufacturer (eBioscience). Total serum IgE was measured using the following reagents from BD: anti-mouse IgE capture antibody (553413), mouse IgE standard (557080), and anti-mouse IgE biotinylated detection antibody (553419). HDM and OVA-specific antibodies were analyzed by enzyme-linked immunosorbent assay in Nunc-Immuno plates which had been coated overnight at 4 °C with 100 μl HDM (25 μg/ml in PBS) or OVA (20 μg/ml in PBS) and blocked for 1 h at 25 °C with assay diluent (eBioscience). Wells were washed several times and mouse serum samples diluted in assay diluent were added for 2 h or overnight. Wells were washed and then biotinylated anti-mouse immunoglobulin IgG1a (553441; BD) or IgG2a (553455; BD) diluted to a concentration of 0.5 μg/ml in assay diluent was added for 1 h. Wells were washed and then streptavidin-conjugated HRP (eBioscience) was added for 30 min. Wells were washed seven times, and then incubated for 15 min with tetramethylbenzidine substrate, or until the reaction approached saturation. Linear regression analysis or absorbance levels were used to determine antibody titers or relative differences.