The ALLPreT panel of legume allergen preparations: characterization of optimized protein extracts for allergen composition, IgE-binding and IgE-crosslinking properties.

INTRODUCTION: Allergenicity risk assessment is essential for the authorization of novel foods in accordance with EU Regulation (EU) 2015/2283. Many published studies on the allergenicity assessment of novel foods have been based on study-specific protein preparations with limited comparability across studies. The study aim was to optimize, harmonize and characterize protein extraction from legumes to enable consistent isolation of allergens from relevant protein families in legumes and to be as compatible as possible with in vitro cell assays and in vivo animal models.

METHODS: Various common (peanut, soybean, lupine, pea, lentil, chickpea, white bean, field bean) and small-seeded (fenugreek, alfalfa, red and white clover) legumes were extracted using 4 different buffers at pH ranging from 7.1 to 9.6. The extracts were prepared using suspension, incubation, centrifugation, filtration, and lyophilization processes. Optimized extracts were characterized for their protein profile (SDS-PAGE), protein composition (mass spectrometry), IgG and IgE-binding properties (Western blot), and functional IgE-crosslinking capacity (RBL-2H3 cell mediator release assay, MRA).

RESULTS: Ammonium carbonate buffer (pH 9.0), of which all buffer components fully evaporate during a post-extraction freeze-drying step, resulted in high protein yield with a balanced protein pattern of allergenic proteins within important protein families [11S, 7S, 2S storage proteins; seed albumin, pathogenesis related- 10 (PR-10) protein, profilin, lipid transfer protein, oleosin, defensin, cyclophilin, annexin, seed biotinylated protein, late embryogenesis abundant protein, and others]. All extracts bound IgG from polyclonal peanut or soybean protein antisera, and IgE from legume sensitized subjects. All extracts presented functional IgE-crosslinking properties in legume-specific MRA after passive sensitization with allergen-specific human IgE.

DISCUSSION: The developed procedure harmonizes protein extraction from various legumes. The lyophilized extracts preserve allergenic proteins and, due to lack of buffer components, are compatible for allergenicity testing in both cellular and animal-based models, and diagnostic IgE screening assays. This protocol supports standardization in the allergenicity risk assessment of novel legume foods.