Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.
Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions.
Peng
Z
Z
Department of Pediatrics and Child Health, and Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 3P5, Canada. zpeng@ms.umanitoba.ca
Xu
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W
James
A A
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Lam
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Sun
D
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Cheng
L
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Simons
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FE
D000330:Aedes / Q000276:immunology*; D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D052179:Antigens, Plant; D016367:Baculoviridae / Q000235:genetics; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D001733:Bites and Stings / Q000276:immunology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006968:Hypersensitivity, Delayed / Q000175:diagnosis / Q000276:immunology; D006969:Hypersensitivity, Immediate / Q000175:diagnosis / Q000276:immunology; D019476:Insect Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007526:Isoelectric Point; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012882:Skin Tests; D018411:Spodoptera / Q000235:genetics; D013997:Time Factors
C446620:Aed a 1 allergen; D000485:Allergens; D052179:Antigens, Plant; D019476:Insect Proteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides