Purified mouse ige

For detection of antibodies in serum, plates were coated with rat-anti-mouse IgE (R35-72; BD) or goat-anti-mouse IgG (Southern Biotech) overnight at 4°C. Purified mouse IgE (BD) or unlabelled mouse IgG1, IgG2a or IgG2b (all Southern Biotech) were used to generate a standard curve. Goat-anti-mouse IgE-AP, IgG1-AP, IgG2a-AP or IgG2b-AP (all Southern Biotech) were used for detection. For detection of parasite-specific antibodies, plates were coated with 20 μg/mL of HES suspension overnight at 4°C. After blocking for 2 hrs with 3% BSA, samples were incubated at room temperature for 2 hrs and the AP-coupled antibodies named above were used for detection. Absorption was measured on a Multiscan FC photometer (Thermo Fisher) at 405 nm and blank wells were used to subtract background.

IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM were measured using a kit (Mouse Isotyping Panel 1 kit) from MSD according to their protocol. Plasma samples were diluted 1∶1000, to 1∶125,000 to ensure samples reading within the linear range of the assay for each antibody.
To measure total IgE, standard 96-well MSD plates were coated (overnight, 4°C) with anti-IgE (2 µg/ml, PBS, BD). All further incubations were carried out at room temperature on an orbital shaker. Unbound antibody was removed by washing (0.05% Tween 20 in PBS), and non-specific binding blocked (1% BSA/PBS, 200 µl/well) for 1 h. The plate was washed, 25 µl standard (14–10,000 ng/ml, purified mouse IgE, BD) or sample (diluted 1∶10) added and incubated for 2 h. Unbound antibody was removed by washing and 25 µl biotinylated anti-mouse IgE (1 µg/ml; BD) pipetted per well. Following 1 h incubation, the plates were washed and 25 µl streptavidin Sulfo-Tag (1 µg/ml; MSD) added for a further 1 h. Plates were then washed, 150 µl Read buffer (T- x2; MSD) added per well and the samples analysed on SI6000 (MSD). OVA-specific IgE was measured by ELISA (AbD Serotec) according to their protocol.