IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2.
10.4168/aair.2015.7.4.376
- Author:
Mey Fann LEE
1
;
Chia Wei CHANG
;
Pei Pong SONG
;
Guang Yuh HWANG
;
Shyh Jye LIN
;
Yi Hsing CHEN
Author Information
1. Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
- Publication Type:Original Article
- Keywords:
Cockroach allergy;
IgE-binding epitope;
synthetic peptide;
specific immunotherapy;
Per a 2
- MeSH:
Amino Acid Sequence;
Cockroaches;
DNA Primers;
DNA, Complementary;
Enzyme-Linked Immunosorbent Assay;
Epitope Mapping*;
Epitopes;
Epitopes, B-Lymphocyte;
Feces;
Humans;
Hypersensitivity;
Immunoblotting;
Immunoglobulin E;
Immunohistochemistry;
Mouth;
Peptides;
Periplaneta*;
Polymerase Chain Reaction;
Protein Sorting Signals;
Taiwan
- From:Allergy, Asthma & Immunology Research
2015;7(4):376-383
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: Cockroaches 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. METHODS: The 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. RESULTS: Human 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. CONCLUSIONS: Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.
