D3.103 - Comprehensive Allergen Characterization for Standardized Basophil Activation Testing

In vitro cellular assays for food allergy diagnosis measure the activation of effector cells upon exposure to the culprit allergen. These assays have emerged as highly specific diagnostic tools capable of distinguishing simple sensitization from true clinical allergy. Among them, the basophil activation test (BAT)—which quantifies allergen‑induced activation of peripheral blood basophils—has been incorporated into EAACI guidelines and helps reduce the need for high‑risk oral food challenges. To ensure consistent quality and reproducibility across laboratories, standardization is required not only for BAT protocols but also for the allergen preparations used. Here, we present a comprehensive characterization of egg allergens and their optimization for use with the IVDR‑compliant Flow CAST BAT kit, addressing a key diagnostic need in pediatric egg allergy, one of the most common food allergies in children.

To develop standardized allergens suitable for BAT, different egg extracts were carefully analyzed using SDS-PAGE (Coomassie staining), LC-MS and IgE-binding assays to evaluate their integrity and composition with particular focus on key allergens. Due to the lack of fresh allergic blood, the capacity of the extracts to elicit cellular responses was tested the using indirect Basophil Activation Test (iBAT), which uses basophils from non-allergic blood donors that are sensitized through the incubation with serum or plasma from allergic individuals.

SDS-PAGE with Coomassie staining and LC-MS were instrumental in assessing the sample integrity and identifying the protein content, allowing the relative quantification of the most important allergens. Anti-IgE ELISA enabled efficient evaluation of IgE‑binding capacity; however, the most informative method was iBAT, which could be used to generate dose-response curves comparable to whole blood BAT, supporting its suitability for allergen optimization and batch‑to‑batch consistency assessment.

Here, we show an extended characterization of the egg allergens. The presence of important allergens in the extract ensures that BAT can correctly identify allergic patients, allowing precise diagnosis. Moreover, our data highlight the value of iBAT as a reliable tool for allergen development, strengthening ongoing efforts toward standardized BAT reagents across laboratories.