title: Allergenicity assessment of apple cultivars: hurdles in quantifying labile fruit allergens.
abstract: BACKGROUND
  Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes.
  
  
  METHODS
  Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS).
  
  
  RESULTS
  Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by approximately 1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen.
  
  
  CONCLUSIONS
  Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.
authors.affiliation: Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Zuidmeer
authors.forename: W A
authors.initials: WA
authors.lastname: van Leeuwen
authors.forename: I
authors.initials: I
authors.lastname: Kleine Budde
authors.forename: H
authors.initials: H
authors.lastname: Breiteneder
authors.forename: Y
authors.initials: Y
authors.lastname: Ma
authors.forename: C
authors.initials: C
authors.lastname: Mills
authors.forename: A I
authors.initials: AI
authors.lastname: Sancho
authors.forename: E J
authors.initials: EJ
authors.lastname: Meulenbroek
authors.forename: E
authors.initials: E
authors.lastname: van de Weg
authors.forename: L
authors.initials: L
authors.lastname: Gilissen
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: R
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000032:analysis* / Q000276:immunology; D052179:Antigens, Plant; D005638:Fruit / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000592:standards; D027845:Malus; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis* / Q000592:standards*; D013045:Species Specificity
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C095551:MALD1 protein, Malus domestica; D010936:Plant Extracts; D010940:Plant Proteins