IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2.
OBJECTIVE
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.
Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.; General Education Center, Tunghai University, Taichung, Taiwan.
Mey Fann
MF
Lee
Department of Life Science, Tunghai University, Taichung, Taiwan.
Chia Wei
CW
Chang
Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
Pei Pong
PP
Song
Department of Life Science, Tunghai University, Taichung, Taiwan.
Guang Yuh
GY
Hwang
School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan.
Shyh Jye
SJ
Lin
Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan.; Department of Life Science, Tunghai University, Taichung, Taiwan.; Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan. ysanne@vghtc.gov.tw.
Yi Hsing
YH
Chen
Cockroach allergy; IgE-binding epitope; Per a 2; specific immunotherapy; synthetic peptide