Production and characterization of an allergen panel for component-resolved diagnosis of celery allergy
Merima Bublin
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorStefano Alessandri
Consorzio Interuniversitario Risonanze, Magnetiche di Metalloprotiene Paramagnetiche and Department of Agricultural Biotechnology, University of Florence, Florence, Italy
Search for more papers by this authorPeter Briza
Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Search for more papers by this authorChristian Radauer
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorMartin Himly
Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Search for more papers by this authorHeimo Breiteneder
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorMerima Bublin
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorStefano Alessandri
Consorzio Interuniversitario Risonanze, Magnetiche di Metalloprotiene Paramagnetiche and Department of Agricultural Biotechnology, University of Florence, Florence, Italy
Search for more papers by this authorPeter Briza
Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Search for more papers by this authorChristian Radauer
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorMartin Himly
Department of Molecular Biology, University of Salzburg, Salzburg, Austria
Search for more papers by this authorHeimo Breiteneder
Department of Pathophysiology, Medical University of Vienna, Vienna, Austria. Fax: +43-1-404005130
Search for more papers by this authorAbstract
In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future.
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