Allergens and Allergen-specific monoclonal antibodies
The production of recombinant Fel d 1 and monoclonal Fel d 1-specific IgE F127 antibody used in these experiments have been described previously.
The anti-Ara h2 IgE sequence (P12P3D08) was obtained from Croots et al. It was expressed as mouse IgE and produced in HEK cells using Expifectamine® (Thermo Fisher Scientific, Waltham, MA, USA) and purified by immunoaffinity using a CaptureSelect XP C‐tag column (Thermo Fisher).
Roasted peanut extract (Ara R) was prepared according to the protocol of Koppelman et al.
Antibodies were deglycosylated using PNGase F deglycosylation kit (Thermo Fisher) under native conditions according to the supplier’s protocol, except that the protein was deglycosylated at 37°C and overnight. The antibodies were purified with Zeba™ Spin Desalting Columns (MWCO 40K MWCO; Thermo Fisher). PNGase F treated IgE is hereinafter called IgE(PNG).