title: Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy.
abstract: Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.
authors.affiliation: Deutsche Forschungsanstalt für Lebensmittelchemie, Garching, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gruber
authors.forename: Martin
authors.initials: M
authors.lastname: Suhr
authors.forename: Andreas
authors.initials: A
authors.lastname: Frey
authors.forename: Wolf-Meinhard
authors.initials: WM
authors.lastname: Becker
authors.forename: Thomas
authors.initials: T
authors.lastname: Hofmann
mesh_terms: D055516:2S Albumins, Plant; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology; D052179:Antigens, Plant; D010367:Arachis; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D008401:Gas Chromatography-Mass Spectrometry; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021183:Peanut Hypersensitivity / Q000276:immunology*; D010940:Plant Proteins; D011817:Rabbits; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D000941:Antigens; D052179:Antigens, Plant; C078642:Ara h II protein, Arachis hypogaea; D000939:Epitopes; D006023:Glycoproteins; D007106:Immune Sera; D010940:Plant Proteins; D007073:Immunoglobulin E