Mejora del diagnóstico in vitro de IgE específica frente a los alérgenos inhalantes Fra e 1, Sal k 1 y Lep d 2 para el análisis de muestras séricas

Immunoglobulin E-mediated allergy or type I hypersensitivity affects a high percentage of the population worldwide, mainly to industrialized and developed countries, causing enormous cost for society. Identifying specific IgE (sIgE) antibodies against allergens from the serum of the patients has a fundamental role in in vitro diagnosis of allergies and in the selection of a proper Allergen Immunotherapy (AIT) treatment. Automatized analysis on ImmunoCAP-250 platform are used to quantify the concentration of sIgE of the serum by immunoassays. Nowadays not every allergen from an allergen source are available for molecular diagnosis, therefore it is necessary to develop diagnosis methods based on purified allergens or allergenic extracts. The aim of this project is to set the appropriate working conditions to quantify the levels of sIgE against the major allergens from Fraxinus excelsior (Fra e 1), Salsola kali (Sal k 1) and Lepidoglyphus destructor (Lep d 2) in ImmunoCAP-250 platform. Biotinylation of allergens Fra e 1, Sal k 1 and Lep d 2 was done for its coupling to the streptavidin solid phase (CAP). Allergen concentration for binding to CAP was optimized. This procedure allowed measuring the sIgE concentration from Fraxinus excelsior, Salsola kali and Lepidoglyphus destructor allergic patients. The project was complemented with a correlation analysis between the sIgE levels against molecular components and the sIgE levels against whole allergenic extract. Antigen-antibody reaction specificity was studied by inhibition experiments carried out using lyophilized whole allergenic extracts. The inhibition experiments also were carried through to verify the cross-reactivity produced between the allergens Fra e 1 and Ole e 1, which comes from Olea europaea. The results suggest that concentration for coupling to CAP depends on the allergen used. A significant strong correlation was obtained between sIgE against molecular components and whole allergenic extract (r ≥ 0,8755) with the exception of sIgE against Lep d 2 and L. destructor which showed a less relevant correlation (r = 0,6852). Fraxinus excelsior allergic patients showed similar values of sIgE against Fra e 1 and against Ole e 1 due to cross-reactivity caused by its homology In conclusion, the developed sIgE diagnosis method is effective to diagnose Fraxinus excelsior, Salsola kali and Lepidoglyphus destructor allergic patients on ImmunoCAP-250 assays