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 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). 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.

• MeSH
• alergeny chemie   imunologie   izolace a purifikace   MeSH
• alergie na pšenici diagnóza   imunologie   MeSH
• atopická dermatitida diagnóza   imunologie   MeSH
• bazofily imunologie   MeSH
• dítě MeSH
• dospělí MeSH
• imunoglobulin E krev   MeSH
• isoelektrická fokusace MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mouka analýza   MeSH
• potravinová alergie diagnóza   imunologie   MeSH
• předškolní dítě MeSH
• pšenice chemie   imunologie   MeSH
• rostlinné proteiny chemie   imunologie   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• ultrafiltrace MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH