C38 2

Serum was separated by centrifugation and stored at −80 °C until analysis. Total IgE levels were determined using ELISA. Purified rat anti-mouse IgE capture mAb (R35-72, BD Biosciences) was used to coat the plates. Purified mouse IgE (C38-2, BD Biosciences) served as a standard, and biotinylated rat anti-mouse IgE (R35-92, BD Biosciences) was used for IgE detection.

K14.E7 mice and non-transgenic littermates were bled at 14-16 weeks of age. Sera were collected and stored at −20°C. Total serum IgE was measured by ELISA as described ^{72 (link)}. Briefly, An ELISA plate (Nunc MaxiSorp) was coated with purified anti-mouse IgE (BD Pharmingen clone R35-72, 1.25 μg/ml, 100 μl/well) in carbonate buffer pH9.2 at 4°C. Wells were washed and blocked with 200 μl of PBS 10% FCS. Purified mouse IgE isotype control (BD Pharmingen C38-2, 0.5mg/ml) antibody was used to create a standard curve, starting at 1000ng/ml. 100 μl/well of sera was incubated for 2h at room temperature. Biotin rat anti-mouse IgE was used as a detection antibody (BD Pharmingen clone R35-118, 1.25 μg/ml, 100 μl/well) and detected using a BD OptiEA Streptavidin HRP (BD Biosciences cat. 51-9002813, 100μl/well), and BD TMB substrate (BD Biosciences cat. 555214, 100μl/well). The plate was read at 450nm using a Synergy HT multi-mode plate reader (BioTek).