Biotin rat anti mouse ige

Based on the antigen used for immunizations, NP conjugated to BSA at a >20 ratio (Biosearch Technologies), PE or OVA all diluted in PBS were coated onto separate 96 well plates (Greiner Bio-one) at 2 μg/mL and incubated overnight at 4 ^°C. Plates were washed then blocked with casein (Thermo Scientific) for one hour at room temperature (RT). Serum samples were serially diluted in casein then added to the plates and incubated for two hours at RT. After washing, biotin rat anti-mouse IgG1 (BD), peroxidase conjugated goat anti-mouse IgM (Jackson Immunoresearch) or biotin rat anti-mouse IgE (BD) was added to the plates and incubated for one hour at RT. Plates were washed and streptavidin HRP (Jackson Immunoresearch) was added to the IgG1 and IgE plates and incubated for thirty minutes at RT. Plates were washed then developed for twenty minutes with an o-Phenylenediamine dihydrochloride tablet (Sigma) dissolved in 100 mM Sodium phosphate dibasic (Mallinckrodt), 50 mM citric acid (Mallinckrodt) and 0.04% hydrogen peroxide (Mallinckrodt) at pH 5.0. Reactions were stopped using 1N HCL (Fisher Scientific) and plates were read at 490λ using a Molecular Devices Spectra Max 340 PC running Soft Max Pro 3.1.2. All antibody data are presented at IgM (1:1000), IgG1 (1:80,000), and IgE (1:100) serum dilutions which represent non-saturating concentrations.

Total serum IgE was assessed using OptEIA ELISA kits (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions. An indirect ELISA method was used to assess the HDM-specific IgE and IgG1 levels in serum samples. Briefly, 96-well microtiter plates were coated overnight with 100㎍/mL HDM in PBS. The next day, 200㎕ of blocking solution (1% BSA in PBS) was added to the plate before the addition of serum samples that had been diluted 1:200 (for HDM-specific IgG1) in blocking buffer, or undiluted (for HDM-specific IgE) for 1h. Subsequently, 100㎕ of sample was added to the plate for 2 h, followed by biotin-rat-anti-mouse IgE or biotin-rat-anti-mouse IgG1 (2㎍/ml; BD Biosciences, San Diego, CA) for 1 h. To detect biotin-labeled IgE or IgG1, streptavidin-HRP (1:100, R&D Systems, Minneapolis, MN) was added to the plate for 30 min. Next, a tetramethylbenzidine substrate reagent set (1:1, BD Bioseicences) was added to detect levels of IgE or IgG1.

Cytokine concentrations were measured in cell culture supernatants using either FlowCytomix (eBioscience) or a LegendPlex Mouse Th1/Th2 Panel (BioLegend) flow cytometry multi-analyte detection system for IL-4, IL-2, IL-5, and IL-13 per the manufacturer’s instructions. Serum IgE (and purified mouse IgE standard; BD) was captured overnight on a plate coated with 2 µg/ml rat anti–mouse IgE (R35-72; BD) and detected with biotin rat anti–mouse IgE at 1 µg/ml (R35-118; BD), streptavidin HRP (BD), and ABTS One Component HRP Microwell substrate (SurModics). H. polygyrus antigen (HEX) was obtained by homogenizing adult worms in PBS. Serum antigen-specific IgG1 was captured on a plate coated with 5 µg/ml HEX and detected using biotin rat anti–mouse IgG1 (Invitrogen), streptavidin HRP, and ABTS.

Total IgE levels were determined by coating plates with 1 μg/ml purified rat anti–mouse IgE (R35–72; BD Biosciences). Standard curve was prepared with purified mouse IgE κ (C38–2; BD Biosciences) and bound Abs in serum samples were detected with biotin rat anti–mouse IgE (R35–118; BD Biosciences) and streptavidin–alkaline phosphatase (Invitrogen). Alkaline phosphate substrate (Moss, Inc.) was added, and color development was detected with SpectraMax® M2 (Molecular Devices) at 405 nm.