Rat anti mouse igg1 clone a85 1

Each incubation step in the ELISAs described below is followed by 3–5 washing steps using PBS containing 0.05% Tween-20. For detection of HDM-specific serum antibodies, MaxiSorp ELISAs plates (Nunc) were coated with 5 μg ml⁻¹ HDM at 4 °C overnight, followed by blocking with 1% bovine serum albumin in PBS for at least 2 h at room temperature. Sera diluted in PBS containing 1% bovine serum albumin were added and incubated in the blocked wells for 2 h at 37 °C. We detected bound IgG₁ and IgE antibodies using biotinylated detection antibodies (rat anti-mouse IgG₁ (clone A85-1, BD Pharmingen; incubated for 1 h at room temperature) and rat anti-mouse IgE (clone R35-118, BD Pharmingen), respectively), followed by incubation with horseradish peroxidase-conjugated streptavidin (BD Pharmingen) for 30 min at room temperature and detection using supersensitive 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma). Antibody titers were calculated by plotting the serum dilution that gave half-maximal signal of a reference serum. Measurements of mediators from BAL fluid was performed as described above.