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Development of an epitope‐specific analytical tool for the major peanut allergen Ara h 2 using a high‐density multiple‐antigenic peptide strategy
Author(s) -
Gruber Patrick,
Suhr Martin,
Frey Andreas,
Becker WolfMeinhard,
Hofmann Thomas
Publication year - 2004
Publication title -
molecular nutrition and food research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.495
H-Index - 131
eISSN - 1613-4133
pISSN - 1613-4125
DOI - 10.1002/mnfr.200400005
Subject(s) - epitope , linear epitope , allergen , epitope mapping , chemistry , edman degradation , antiserum , antigen , peptide , immunoglobulin e , microbiology and biotechnology , peptide sequence , biochemistry , antibody , biology , immunology , allergy , gene
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 TM 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.