A new approach to the isolation and characterization of wheat flour allergens

Abstract

The incidence of food allergy to wheat is increasing. Its diagnosis depends on the purity of major allergens and their inclusion in tests. Isolation and characterization of wheat allergens are therefore of utmost importance. To purify and identify wheat flour allergens most frequently recognized by patients' IgE antibodies and to study their allergenicity. Water/salt-soluble extracts from wheat flour were prepared and separated using a combination of ultrafiltration, isoelectric focusing and liquid chromatography. Purified proteins were analysed by immunoblotting using pooled sera from patients with atopic dermatitis who possessed IgE specific to wheat. Wheat proteins found to bind IgE were subsequently identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The frequency and intensity of IgE binding of isolated proteins were tested using individual sera from patients and controls. We developed a procedure that allows isolation of wheat allergens from natural sources. Twenty-seven potential wheat allergens have been successfully identified; of these, the following seven are newly reported in food allergy: endogenous α-amylase/subtilisin inhibitor, trypsin/α-amylase inhibitor (AAI) CMX1/CMX3, thaumatin-like protein (TLP), xylanase inhibitor protein-1, β-glucosidase, class II chitinase and 26 kDa endochitinase. TLP and wheatwin were shown to activate patients' basophils to a similar extent as two well-known allergens, lipid transfer protein (Tri a 14) and AAI 0.19 (Tri a 28.0101). Our new approach enables the isolation of water/salt-soluble wheat allergens in their native form in amounts sufficient both for biological testing (in vivo and in vitro) and for physicochemical characterization. Such studies will lead to a more detailed knowledge of allergenicity of wheat proteins and to improved accuracy of diagnostic tests.