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.
CITATION STYLE
Morales, S., Castro, A. J., Jimenez-Lopez, J. C., Florido, F., Rodríguez-García, M. I., & De Dios Alché, J. (2012). A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens. Electrophoresis, 33(9–10), 1367–1374. https://doi.org/10.1002/elps.201100667
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