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Morales S.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas CSIC | Morales S.,Hospital Universitario San Cecilio | Castro A.J.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas CSIC | Jimenez-Lopez J.C.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas CSIC | And 3 more authors.
Electrophoresis | Year: 2012

Standardization of pollen protein extracts is essential in order to ensure efficiency and safety in allergy diagnosis and immunotherapy. In this paper, we have optimized a multiplex Western blotting method for the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5, and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen-protein samples from six olive cultivars. In addition, we also tested the IgE-binding capacity of these pollen extracts by reprobing the same blot with a pool of sera from eight patients allergic to olive and detection with enzyme conjugated antibodies. A noticeable variability regarding allergen content and IgE-reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the IgE-binding proteins by simply overlapping both fluorescence and chemiluminescence images. This method is versatile since it can be applied to other allergogenic plant species and extended to other allergens. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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