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OPTIMALIZATION OF EPITOPE EXTRACTION TECHNIQUE FOR EPITOPE MAPPING OF FOOD ALLERGEN

Autoři: Ouzká Šárka | Bílková Zuzana | Křenková Jana | Královský Josef

Rok: 2005

Druh publikace: ostatní - přednáška nebo poster

Název zdroje: 11th International Symposium on Separation Sciences 2005

Název nakladatele: University of Pardubice

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Jazyk	Název	Abstrakt	Klíčová slova
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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