Diseño y aplicaciones de un sistema multiplex para la detección y caracterización de proteínas alergénicas en el polen del olivo (olea europaea l.)

Resumen

Olive pollen is released in huge amounts at the end of the spring season, representing a highly significant cause for seasonal allergy. The protein responsible for these phenomena are the allergens named Ole e 1 to Ole e 11, which play key functions in the physiology of the pollen grain. In this work, we have optimized a system for multiple detection of four of these allergens (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) in different analytical systems including membrane immunodetection (western blotting), confocal laser scanning microscopy and transmission electron microscopy. This method has been used to characterize the allergen profiles of the pollen grains in a wide sample of the germplasm of this species, and to analyze their connection with several physiological and agronomical parameters, including the ability to germinate in vitro, the self-incompatible character and flowering phenology of the origin cultivar. Moreover, correlations between the allergen expression profiles and climate parameters have been established. This study includes the expression analysis of the four allergens mentioned above over in vitro pollen germination, and their simultaneous localization in the pollen grain and the pollen tube, both by confocal microscopy and at the ultrastructural level. Finally, practical considerations have been made addressed to make compatible the extreme allergen variability present in the nature and the personalization of the extracts prepared for clinical purposes (diagnosis and immunotherapy), by establishing cultivars with model allergen profiles.