Development and validation of a mouse-based primary screening method for testing relative allergenicity of proteins from different wheat genotypes

Background: Wheat allergy is a major food allergy that has reached significant levels of global public health concern. Potential variation in allergenicity among different wheat genotypes is not well studied at present largely due to the unavailability of validated methods. Here, we developed and validated a novel mouse-based primary screening method for this purpose. Methods: Groups of Balb/c mice weaned on-to a plant protein-free diet were sensitized with salt-soluble protein (SSP) extracted from AABB genotype of wheat (durum, Carpio variety). After confirming clinical sensitization for anaphylaxis, mice were boosted 7 times over a 6-month period. Using a pooled-plasma mini bank, a wheat-specific IgE-inhibition (II)-ELISA was optimized. Then the relative allergenicity of SSPs from tetraploid (AABB), hexaploid (AABBDD) and diploid (DD) wheat genotypes were determined. The IC ₅₀ /IC ₇₅ values were estimated using IgE inhibition curves. Results: The optimized II-ELISA with an inhibition time of 2.5 h had a co-efficient of variation of <2%. Primary screening for relative allergenicity demonstrated that IgE binding to AABB-SSP was significantly abolished by the other two wheat genotypes. Compared to AABB, the relative allergenicity of SSPs of AABBDD and DD were significantly lower (p <.01). Furthermore, IgE inhibition curves showed significant differences in IC ₅₀ and IC ₇₅ values among the three wheat genotypes. Conclusion: We report a novel mouse-based primary screening method of testing relative allergenicity of wheat proteins from three different wheat genotypes for the first time. This method is expected to have broad applications in wheat allergy research.