title: Lipid transfer proteins from fruit: cloning, expression and quantification.
abstract: BACKGROUND
  Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs.
  
  
  METHODS
  cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3.
  
  
  RESULTS
  The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3.
  
  
  CONCLUSIONS
  rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.
authors.affiliation: Department of Immunopathology, Sanquin, Amsterdam, The Netherlands.
authors.forename: Laurian
authors.initials: L
authors.lastname: Zuidmeer
authors.forename: W Astrid
authors.initials: WA
authors.lastname: van Leeuwen
authors.forename: Ilona Kleine
authors.initials: IK
authors.lastname: Budde
authors.forename: Jessica
authors.initials: J
authors.lastname: Cornelissen
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bulder
authors.forename: Ilona
authors.initials: I
authors.lastname: Rafalska
authors.forename: Noèlia Telléz
authors.initials: NT
authors.lastname: Besolí
authors.forename: Jaap H
authors.initials: JH
authors.lastname: Akkerdaas
authors.forename: Riccardo
authors.initials: R
authors.lastname: Asero
authors.forename: Montserrat
authors.initials: M
authors.lastname: Fernandez Rivas
authors.forename: Montserrat Fernandez
authors.initials: MF
authors.lastname: Rivas
authors.forename: Eloina
authors.initials: E
authors.lastname: Gonzalez Mancebo
authors.forename: Eloina Gonzalez
authors.initials: EG
authors.lastname: Mancebo
authors.forename: Ronald
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D019976:Cloning, Organism; D005638:Fruit / Q000276:immunology*; D027845:Malus / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins; D014176:Protein Biosynthesis; D027861:Prunus / Q000276:immunology*; D011817:Rabbits
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; C463180:Mal d 3 allergen; D010940:Plant Proteins; C463181:Pru p 3 allergen; C511920:lipid transfer proteins, plant