title: The major grass pollen group 5 allergen from Dactylis glomerata and its C-terminal split product both behave as dimers: implications for allergen standardization.
abstract: BACKGROUND
  On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M(r)) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M(r). The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays.
  
  
  METHODS
  Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity.
  
  
  RESULTS
  The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD(L)/(A)GY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE.
  
  
  CONCLUSIONS
  These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.
authors.affiliation: Sanquin, Department of Immunopathology and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Erica
authors.initials: E
authors.lastname: van Oort
authors.forename: Miranda C
authors.initials: MC
authors.lastname: Dieker
authors.forename: Pleuni G
authors.initials: PG
authors.lastname: de Heer
authors.forename: Gabriel
authors.initials: G
authors.lastname: Peltre
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: Ronald
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000276:immunology*; D052179:Antigens, Plant; D031745:Dactylis / Q000276:immunology*; D019281:Dimerization; D007118:Immunoassay; D010940:Plant Proteins / Q000276:immunology*; D011058:Pollen / Q000276:immunology*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C444200:Dac g 5 allergen; D010940:Plant Proteins