IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2 |
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Authors: | Mey-Fann Lee Chia-Wei Chang Pei-Pong Song Guang-Yuh Hwang Shyh-Jye Lin Yi-Hsing Chen |
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Institution: | 1.Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.;2.Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan.;3.General Education Center, Tunghai University, Taichung, Taiwan.;4.Department of Life Science, Tunghai University, Taichung, Taiwan.;5.School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan.;6.Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan. |
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Abstract: | PurposeCockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach.MethodsThe cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry.ResultsHuman linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces.ConclusionsInformation on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy. |
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Keywords: | Cockroach allergy IgE-binding epitope synthetic peptide specific immunotherapy Per a 2 |
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