title: Peanut protein extraction conditions strongly influence yield of allergens Ara h 1 and 2 and sensitivity of immunoassays.
abstract: The clinical importance of peanut (Arachis hypogaea) allergies demands standardized allergen extraction protocols. We determined the effectiveness of common extraction conditions (20 buffers, defatting reagents, extraction time/temperatures, processing, extraction repeats) on crude protein and Ara h 1 and 2 yields. Despite similar 1D-gel profiles, defatting with n-hexane resulted in significantly higher yields of crude protein, Ara h 1, and Ara h 2 than with diethyl ether. The yields were affected by the composition and pH of the extraction buffers and other conditions, but crude protein yield did not always correlate with Ara h 1 and 2 yields. Denaturants, reducing agents, acidic buffers, and thermal processing of peanuts perturbed allergen quantification in ELISAs, probably via exposure of additional epitopes. Allergen detection in 2D-Western blots with PBS resulted in greater sensitivity than with TBS or Tris. We recommend that allergen extraction conditions be selected based on the research question being investigated.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia; School of Biological Sciences, University of Sydney, Sydney, Australia. Electronic address: walczyk.nicole@gmail.com.
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Walczyk
authors.affiliation: School of Biological Sciences, University of Sydney, Sydney, Australia. Electronic address: penny.smith@sydney.edu.au.
authors.forename: Penelope M C
authors.initials: PMC
authors.lastname: Smith
authors.affiliation: Woolcock Institute of Medical Research, Sydney, Australia. Electronic address: euan.tovey@sydney.edu.au.
authors.forename: Euan R
authors.initials: ER
authors.lastname: Tovey
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia; Plant Breeding Institute, Faculty of Agriculture and Environment, University of Sydney, Sydney, Australia. Electronic address: thomas.roberts@sydney.edu.au.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Roberts
mesh_terms: D000485:Allergens / Q000737:chemistry*; D052179:Antigens, Plant / Q000737:chemistry*; D010367:Arachis / Q000737:chemistry*; D006586:Hexanes; D006801:Humans; D021183:Peanut Hypersensitivity / Q000276:immunology*; D010940:Plant Proteins / Q000737:chemistry
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D006586:Hexanes; D010940:Plant Proteins; C026385:n-hexane
keywords: Allergen; Ara h 1; Ara h 2; ELISA; Extraction; Immunochemistry; Peanut; Western blot