title: Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.
abstract: 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.
authors.affiliation: Department of Pediatrics and Child Health, and Department of Immunology, University of Manitoba, Winnipeg, Manitoba R3E 3P5, Canada. zpeng@ms.umanitoba.ca
authors.forename: Z
authors.initials: Z
authors.lastname: Peng
authors.forename: W
authors.initials: W
authors.lastname: Xu
authors.forename: A A
authors.initials: AA
authors.lastname: James
authors.forename: H
authors.initials: H
authors.lastname: Lam
authors.forename: D
authors.initials: D
authors.lastname: Sun
authors.forename: L
authors.initials: L
authors.lastname: Cheng
authors.forename: F E
authors.initials: FE
authors.lastname: Simons
mesh_terms: 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
chemical_list: C446620:Aed a 1 allergen; D000485:Allergens; D052179:Antigens, Plant; D019476:Insect Proteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides