Biotin ratanti mouse igg1 a85 1

Allergen-specific IgG₁ and IgE plasma levels were measured by
ELISA. Briefly, plates were coated with either OVA or ASP (100 μg/mL)
overnight at 4°C. Blocking was done with 1% BSA in PBS, and all washes
were performed with 0.05% Tween 20 in PBS. Plasma samples were serially diluted
to determine the optimal dilution for IgG₁ and IgE. After 2 hours of
incubation, the biotinylated secondary antibodies, respectively biotin rat
anti-mouse IgG₁ (A85–1; Pharmingen; BD Biosciences, San Jose,
Calif) and biotin rat antimouse IgE (R35–118; Pharmingen, BD
Biosciences), were added for 1 hour, followed by washing and incubation with
streptavidin–horseradish peroxidase (DY998, R&D Systems, Minneapolis,
Minn). Samples were developed by adding tetramethylbenzidine substrate (BD
Biosciences). MCPT1 plasma levels were measured according to the
manufacturer’s instructions (Invitrogen; Thermo Fisher Scientific,
Waltham, Mass). All reactions were stopped with 2N H₂SO₄,
and absorbance was read at 450 nm.

For IFNα ELISA, 2 × 10⁴ splenic pDCs were sort purified and stimulated with CpG-A 2216 (6 μg ml⁻¹) for 16 h. Supernatants were analysed with an IFNα Mouse ELISA Kit (Invitrogen). Total IgE ELISA was performed with a Mouse IgE ELISA Set (BD) and Reagent Set B (BD). For total IgG1 ELISA, plates were coated with 2.5 μg ml⁻¹ of rat anti-mouse IgG1 (A85-3; catalogue (cat.) no. 553445; BD), blocked, detected with biotin rat anti-mouse IgG1 (A85-1; cat. no. 550331; BD; 1:10,000 dilution) and Streptavidin HRP (cat. no. 554066; BD; 1:1,000 dilution). IgG1 standard was purified mouse IgG1 anti-NFIL3 antibody (sc-374451; Santa Cruz Biotechnology).