title: A new approach to the isolation and characterization of wheat flour allergens.
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Microbiology, Academy of Sciences of Czech Republic, Prague, Czech Republic. psotkovsky@gmail.com
authors.forename: P
authors.initials: P
authors.lastname: Sotkovský
authors.forename: J
authors.initials: J
authors.lastname: Sklenář
authors.forename: P
authors.initials: P
authors.lastname: Halada
authors.forename: J
authors.initials: J
authors.lastname: Cinová
authors.forename: I
authors.initials: I
authors.lastname: Setinová
authors.forename: A
authors.initials: A
authors.lastname: Kainarová
authors.forename: J
authors.initials: J
authors.lastname: Goliáš
authors.forename: K
authors.initials: K
authors.lastname: Pavlásková
authors.forename: S
authors.initials: S
authors.lastname: Honzová
authors.forename: L
authors.initials: L
authors.lastname: Tučková
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D001491:Basophils / Q000276:immunology*; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D003876:Dermatitis, Atopic / Q000175:diagnosis / Q000276:immunology; D005260:Female; D005433:Flour / Q000032:analysis*; D005512:Food Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood*; D007525:Isoelectric Focusing; D008297:Male; D008875:Middle Aged; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000737:chemistry / Q000276:immunology*; D014462:Ultrafiltration; D021182:Wheat Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D055815:Young Adult
chemical_list: D000485:Allergens; D010940:Plant Proteins; D007073:Immunoglobulin E