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.

Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases

“…This prokaryotic expression system is still applied for the production of many different recombinant allergens [103, 104, 105, 106, 107, 108]. In more recent years, several allergens were produced in eukaryotic expression systems like the yeast Pichia pastoris [109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127], insect cells [128, 129, 130, 131, 132, 133, 134, 135, 136], and tobacco plants [137, 138]. These systems facilitate post-translational modifications that do not (easily) occur in E. coli , like disulfide-bridge formation, hydroxylation of prolines, glycosylation.…”

Sensibilidad y especificidad de la prueba cutánea por punción con dos concentraciones del extracto estandarizado de Culex quinquefasciatus en niños alérgicos

“…2,14,15 De manera inusitada hubo una coincidencia total entre los resultados cutáneos considerados positivos y negativos en ambos grupos para las dos concentraciones del extracto de Culex quinquefasciatus. El porcentaje de positividad en el grupo de pacientes alérgicos fue superior a 90%; sin embargo, otros autores reportaron que las respuestas positivas se registran en 32%.…”

“…El porcentaje de positividad en el grupo de pacientes alérgicos fue superior a 90%; sin embargo, otros autores reportaron que las respuestas positivas se registran en 32%. 2,14,15 En esta investigación, la alta coincidencia en el resultado de la prueba cutánea nos muestra que la concentración del extracto a 100 UNP/mL puede sustituirse por la de menor concentración sin perder confiabilidad en el diagnóstico de sensibilización al mosquito Culex quinquefasciatus al usar ese método in vivo.…”

Systemic Immediate Allergic Reactions to Arthropod Stings and Bites

“…Although some of the allergens have been characterized [16, 33, 44, 88], only few allergens have been synthesized with recombinant techniques [15]. Accordingly, most investigations have been made with whole-body extracts or extracts from salivary glands.…”