标题：Caracterización de la actividad alergénica y enzimática de extractos somáticos producidos a partir de cultivos in vitro del ácaro Dermatophagoide farinae
摘要：Introduccion: El Dermatophagoides fainae (DF) es un acaro cosmopolita presente en el polvo intradomiciliario. Este acaro es fuente importante de aereo-alergenos desencadenantes de asma y rinitis alergica. Objetivo: El proposito de este trabajo fue la produccion y caracterizacion de extractos alergenicos para uso en diagnostico alergologico y terapia. Metodos: Los extractos se prepararon a partir de cultivos puros in vitro de acaros. Las proteinas del acaro fueron extraidas mediante lisis osmotica. La concentracion de proteinas se determino por microensayo de Bradford y el perfil electroforetico fue identificado por SDS-PAGE. La actividad enzimatica fue evaluada con el estuche comercial Api Zym (Biomeriux R) y la actividad alergenica por inmunoensayo de Dot Blot. Resultados: Se prepararon cinco lotes de extractos. La media del peso seco de material extraido fue 37,04 mg/gramo de acaros ( DE= 11,3) y la media de la cantidad de proteina fue 212,4 ¿Êg/mg de peso seco (DE= 33,3). El perfil SDS-PAGE mostro la presencia de 8 bandas comunes (21-109 kDa). Todos los extractos presentaron actividad enzimatica hidrolasa. Tambien se demostro actividad tripsina, alfa-amilasa y alfa-quimiotripsina, correspondientes a los alergenos de los acaros del grupo 3, 4 y 6 respectivamente. Todos los extractos tuvieron proteinas con capacidad de union a los anticuerpos IgE. Conclusion: Aqui presentamos la produccion de extractos del acaro DF, con actividad alergenica. Estos resultados fundamentaran la produccion a escala de extractos alergenicos de acaros desencadenantes de enfermedades alergicas en el humano, con fines diagnosticos y de aplicacion terapeutica.
其他摘要：Introduction: Dermatophagoides farinae (DF) is a cosmopolitan mite present in the indoor house dust. This mite is an important source of aeroallergens triggering of allergic asthma and rhinitis. The purpose of the present study was the production, and immunochemical and enzymatic characterization of allergenic extracts for use in allergologic diagnosis and therapy. Materials and methods: DF somatic extracts were prepared from pure In vitro culture of the mite from Santa Marta mite fauna, a Caribbean Colombian City. Mite proteins were extracted by osmotic lysis with 0.1 M of NH4HCO3 buffer. The soluble proteins were separated of the cellular debris by centrifugation, dialysis and filtration. Proteins Protein concentration was measured by the Bradford microassay test and the electrophoretic profile was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic activity was evaluated with the commercial Kit, Api Zym (Biomeriux ®). A Dot blot immunoassay was done in order to evaluate allergenic activity of the extracts. Results: Five lots of DF somatic extracts were prepared. The average of dry weight material extract was 37.04 mg/g of mites (SD= 11.3) and the average of total protein obtained was 212.4 ìg/mg of dry weight (SD= 33.3). The SDS-PAGE profile showed 8 common bands (21-109 kDa). All extracts presented enzymatic activity, esterase (C4), esterase lipase, alkaline phosphatase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, leucine and valine arylamidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and alpha mannosidase. Furthermore, enzymatic activity for trypsin, alpha-amylase and alpha- chymotrypsin, corresponding to the mites allergen groups 3, 4 and 6, respectively, was demonstrated. All somatic DF extracts have IgE antibody binding proteins. Conclusions: We report the production of somatic extracts DF, with allergenic activity. Mite cultures in the exponential growth phase expressed the major mite allergens, trypsin, alpha-amylase and alpha-chymotrypsin. These results would could be the base for the scale production of allergenic extracts of mites responsible for the induction of human allergic diseases, useful for diagnostic and therapeutic applications.
