Anti ige

Total mouse IgE was determined by sandwich-ELISA using kit BD OptEIA ELISA Set (BD, San Diego, CA, USA). OVA-specific IgE was determined by adding serum at multiple dilutions to plates with anti-IgE (SouthernBiotech, Birmingham AL, USA). After washing, biotin-labeled OVA was added and revealed with avidin-HRP plus substrate. Internal sample arbitrarily assigned as 1,000 U was used as standard. OVA-specific IgG2a/c were measured by coating the plates with 20 µg/mL of OVA. Serum samples were added at multiple dilutions and revealed with goat anti-mouse IgG2a conjugated to HRP (Invitrogen, San Diego, CA, USA), which also reacts with IgG2c isotype. Purified mouse IgG2a (Southern Biotech) was used as standard. All ELISA were performed in 96-well maxisorp plates (Nunc, NY, USA). Cytokines levels were assayed by sandwich kit ELISA according to the manufacturer’s recommendation (BD Biosciences, PharMingen, San Diego, CA, USA) as previously described (5 (link)). Values were expressed as picograms per milliliter deduced from a standards curve of recombinant cytokines ran in parallel. The limits of detection ranged from 5 to 30 pg/mL.

ELISA 96‐well plates were coated with HDM or CR (2 μg/mL in PBS) overnight at 4°C and were then washed and blocked with 4% BSA/PBS (Sigma, Poole, Dorset, UK) for 1 h at 37°C. Serum was initially diluted 1/50 and then serially threefold 8 times prior to incubation in allergen‐coated plates at 37°C for 1 h. Horseradish peroxidase (HRP)‐conjugated anti‐IgE (anti‐mouse IgE‐HRP – 1 : 1000) and anti‐IgG1 (anti‐mouse IgG1‐HRP – 1 : 10 000) (antibodies from Southern Biotech, Birmingham, AL, USA) were then added and left for a further hour at 37°C to detect the presence of HDM/CR‐specific antibodies. Samples were developed by addition of KPL SureBlue, TMB microwell peroxidase substrate and reactions terminated by stop solution, 2N H₂SO₄. The final reactions were measured at 405 nm by employing an ELISA microplate reader.