Immunoglobulin immunoassays

For determination of total serum IgE by 2-step sandwich (capture) ELISA, 96-well plates were coated with 2 μg/mL of anti-mouse IgE mAb (BD Pharmingen clone R35-72) in PBS for 2 hours at 37°C. The plates were washed, blocked for 1 hour in PBS/1% BSA, and washed again, after which serum samples or IgE standard (BD Pharmingen, catalog no. 557079) were added in duplicate in PBS/1% BSA overnight at 4°C. For determination of HDM-specific IgG1 and IgG2c by indirect ELISA, 96-well plates were coated overnight at 4°C with 2 μg/mL of HDM extract in PBS (pH 7.2-7.4), washed with 0.05% Tween 20 in PBS, and blocked for 2 hours at room temperature with PBS/1% BSA. The plates were washed, and serum diluted in blocking solution was applied to the wells in triplicate over a series of eight 4-fold dilutions starting at 1:20 and incubated overnight at 4°C. For all isotypes, plates were washed after incubation with samples, and 2 μg/mL of biotinylated isotype-specific secondary antibodies (BD Biosciences) in 1% BSA/PBS were incubated in the plates at room temperature for 2 hours (for IgG2c, a cross-reactive antibody that recognizes IgG2a was used⁴⁷). Plates were washed, and streptavidin-peroxidase (R&D Systems, Minneapolis, Minn) was incubated in the plates at room temperature for 30 minutes. Plates were washed and developed using reagents from R&D Systems; the reaction was stopped with 1N H₂SO₄, and ODs were read by using a Bio-Tek Instruments (Winooski, Vt) Synergy HTX multimode plate reader at 450 nm with background subtraction at 570 nm.