rAed a 4: A New 67-kDa Aedes aegypti Mosquito Salivary Allergen for the Diagnosis of Mosquito Allergy.
BACKGROUND
Accurate diagnosis of mosquito allergy has been hampered by the laborious task of obtaining mosquito salivary allergens. We have previously studied 3 recombinant (r) Aedes aegypti mosquito salivary allergens: rAed a 1, rAed a 2 and rAed a 3. Here, we report the expression, purification, identification and evaluation of rAed a 4, a 67-kDa α-glucosidase.


METHODS
rAed a 4 was expressed using a baculovirus/insect cell system, purified by a combination of anion- and cation-exchange chromatography, and identified by immunoblotting. A. aegypti saliva extract was prepared in our laboratory. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to measure rAed a 4-specific immunoglobulin E (IgE) and IgG antibodies in sera from 13 individuals with a positive mosquito-bite test from a laboratory-reared mosquito. Sera from 18 individuals with a negative bite test served as controls.


RESULTS
Purified rAed a 4 bound to the IgE in mosquito-allergic sera, as detected by ELISA and immunoblotting. The binding of rAed a 4 to IgE could be inhibited in a dose-dependent manner by the addition of an A. aegypti extract. Mosquito-allergic individuals had significantly higher mean levels of rAed a 4-specific IgE and IgG than controls. Using the mean of the controls ± 2 SD as a cut-off level, 46% of the 13 allergic individuals had a positive IgE, while none of the controls was positive (p < 0.001).


CONCLUSIONS
Aed a 4 is a major allergen in mosquito saliva. Its recombinant form has the hydrolase function and can be used for the diagnosis of mosquito allergy.
Departments of Pediatrics and Child Health, University of Manitoba, Winnipeg, Man., Canada.
Zhikang
Z
Peng
Li
L
Caihe
Andrew N
AN
Beckett
Qingdong
Q
Guan
Anthony A
AA
James
F Estelle R
FE
Simons
D000330:Aedes / Q000276:immunology*; D000485:Allergens / Q000276:immunology*; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis* / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007299:Insect Bites and Stings / Q000175:diagnosis* / Q000276:immunology*; D019476:Insect Proteins / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000276:immunology; D012471:Salivary Proteins and Peptides / Q000276:immunology / Q000302:isolation & purification; D012882:Skin Tests
D000485:Allergens; D007074:Immunoglobulin G; D019476:Insect Proteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D007073:Immunoglobulin E