Anti mouse igg2a

ELISA-plates were coated with 5x10⁵ plaque-forming units (PFU) heat-inactivated RSV-A2 in 100 μl carbonate buffer (50 mM carbonate/bicarbonate, pH 9.6) per well overnight at 4°C. Subsequently, the binding sites were blocked with 5% skimmed milk in PBS-T (PBS including 0.05% Tween-20) for one hour at room temperature and the plates were washed with PBS-T. Sera, diluted in 2% skimmed milk in PBS-T, were added to the wells. After 1h incubation at 4°C and three washing steps, secondary detection antibodies were added for 1h at RT: HRP-coupled anti-mouse Ig (1:1000, polyclonal, Daco), anti-mouse IgG1 (1:1000, clone X56, BD Biosciences), anti-mouse IgG2a (1:1000, clone R19-15, BD Biosciences), or anti-mouse IgA (1:5000, polyclonal, Bethyl Laboratories). Plates were washed seven times before ECL solution was added and the signal (relative light units per second, RLU/s) acquired on a microplate luminometer (VICTOR X5, PerkinElmer) using PerkinElmer 2030 Manager software.

Allergen‐specific serum IgG2a levels were measured by ELISA. Briefly, plates were coated with 100 μl of HSE (5 μg/ml) overnight at 4°C and reference wells were coated with antimouse IgG2a (0.5 μg/ml, BD Biosciences Pharmingen). After the wells had been blocked and washed, serum (diluted 1:1000) was added to HSE‐coated wells and purified IgG2a (BD Biosciences Pharmingen) was added to reference wells; the plates were incubated overnight at 4°C. Plates were subsequently washed and incubated with alkaline phosphatase‐labelled goat anti‐mouse IgG2a (diluted 1:2000; Southern Biotechnology Associates) for 2 h at room temperature. After plates had been washed, a solution of 4‐nitrophenyl phosphate (Roche) was added to each well. Absorbance at 450 nm was measured 1 h after the addition of substrate. A standard curve of murine IgG2a was used as a reference.