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OPTIMALIZATION OF EPITOPE EXTRACTION TECHNIQUE FOR EPITOPE MAPPING OF FOOD ALLERGEN
Authors: Ouzká Šárka | Bílková Zuzana | Křenková Jana | Královský Josef
Year: 2005
Type of publication: ostatní - přednáška nebo poster
Name of source: 11th International Symposium on Separation Sciences 2005
Publisher name: University of Pardubice
Place:
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Titles:
Language Name Abstract Keywords
cze Optimalizace techniky "epitopové extrakce" pro epitopové mapování potravinových alergenů určeno pro seznam publikací potravinový alergen; epitopové mapování; enzymový mikroreaktor
eng OPTIMALIZATION OF EPITOPE EXTRACTION TECHNIQUE FOR EPITOPE MAPPING OF FOOD ALLERGEN The specific immunotherapy only with immunogenic peptides (epitopes) of the specific allergen seems to be a good way for therapy of allergy and minimizing of adverse effects incident to the immunotherapy with whole allergen extract. This form of immunotherapy requires identification of major immunogenic allergen epitopes. For this purpose we had chosen the technique of epitope mapping ?Epitope extraction? with combination of newly prepared magnetic enzyme and immunoaffinity reactors and ESI-TOF-MS analysis for a rapid isolation and determination of immunogenic epitopes (peptides) of a model food allergen. We aimed at 1) optimalization of limited and total proteolysis of ovalbumin (model food allergen) using trypsin-reactors and 2) preparation of immunoaffinity reactors (with oriented and/or non-oriented immobilized monoclonal and/or polyclonal anti-ovalbumin antibodies) for isolation of ovalbumin immunogenic peptides. Ovalbumin is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food in children. Due to the resistance of native ovalbumin to proteolytic digestion using enzyme reactors with immobilized trypsin, various conformers of ovalbumin, which could be liable to total and/or limited proteolysis by immobilized trypsin, were prepared and tested (i.e. ovalbumin denaturated only by urea or by urea with dithiotreitol (DTT) and consecutive acetylated by monoiodacetic acid (IAA), S-ovalbumin, I-ovalbumin, deglycosylated ovalbumin). The S-ovalbumin differs from native ovalbumin in its greater stability, compactness, hydrophobicity and in higher resistence to proteolysis whereas denatured ovalbumin using urea, DTT and IAA is the most sensitive ovalbumin conformer. Conditions for the preparation of immunoaffinity reactors with anti-ovalbumin antibodies (type and amount of magnetic carriers, type of immobilization procedure, amount of specific antibody?) were optimized. Next, the binding capacity and stability of these food allergen; epitope mapping; enzyme microreactor

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