BACKGROUND: 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. OBJECTIVE: To purify and identify wheat flour allergens most frequently recognized by patients' IgE antibodies and to study their allergenicity. METHODS: 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. RESULTS: 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 alpha-amylase/subtilisin inhibitor, trypsin/alpha-amylase inhibitor (AAI) CMX1/CMX3, thaumatin-like protein (TLP), xylanase inhibitor protein-1, beta-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). CONCLUSION AND CLINICAL RELEVANCE: 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.
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机译：背景：小麦对食物过敏的发生率正在增加。其诊断取决于主要过敏原的纯度及其在测试中的含量。因此，小麦过敏原的分离和鉴定至关重要。目的：纯化和鉴定患者IgE抗体最常识别的小麦粉过敏原，并研究其致敏性。方法：采用超滤，等电聚焦和液相色谱相结合的方法，制备并分离了小麦粉中的水/盐可溶性提取物。通过使用具有特异于小麦的IgE的特应性皮炎患者的合并血清，通过免疫印迹分析纯化的蛋白质。随后通过基质辅助激光解吸/电离飞行时间质谱法鉴定了发现与IgE结合的小麦蛋白。使用来自患者和对照的个体血清测试分离的蛋白质的IgE结合的频率和强度。结果：我们开发了一种程序，可以从天然来源中分离小麦过敏原。已成功鉴定出二十七种潜在的小麦过敏原。其中，以下7种食物过敏新报道：内源性α-淀粉酶/枯草杆菌蛋白酶抑制剂，胰蛋白酶/α-淀粉酶抑制剂（AAI）CMX1 / CMX3，索马甜蛋白样蛋白（TLP），木聚糖酶抑制剂蛋白-1，β-葡糖苷酶，II类几丁质酶和26 kDa内切几丁质酶。已显示TLP和Wheatwin与两种众所周知的过敏原（脂质转移蛋白（Tri a 14）和AAI 0.19（Tri a 28.0101））活化患者嗜碱性粒细胞的程度相似。结论和临床意义：我们的新方法能够分离天然形式的水/盐溶性小麦过敏原，其数量足以进行生物学测试（体内和体外）和理化特性。这些研究将使人们对小麦蛋白的致敏性有更详细的了解，并提高诊断测试的准确性。
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