Electrophoresis 2012, 33, 1367–1374 1367 Sonia Morales 1*, ** Antonio Jes ´ us Castro 1* Jose Carlos Jimenez-Lopez 1 Fernando Florido 2 Mar´ ıa Isabel Rodr´ ıguez-Garc´ ıa 1 Juan de Dios Alch´ e 1 1 Department of Biochemistry, Cell and Molecular Biology of Plants, Estaci ´ on Experimental del Zaid´ ın, Consejo Superior de Investigaciones Cient´ ıficas (CSIC), Granada, Spain 2 Allergy Service, Hospital Universitario San Cecilio, Granada, Spain Received December 5, 2011 Revised February 19, 2012 Accepted February 20, 2012 Research Article A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens 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 multi- plex 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 detec- tion. 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. Keywords: Allergen / Fab fragment / Multiplex Western blotting / Pollen / Standardization DOI 10.1002/elps.201100667 1 Introduction Olive pollen produces seasonal respiratory allergy in the Mediterranean area and in other temperate climates of the world where this species is intensively cultivated. To date, up to 11 allergenic proteins have been identified and character- ized in olive pollen extracts, called Ole e 1 to Ole e 11 [1–4]. Ole e 1 is the most prevalent sensitizing agent, affecting more than 70% of patients suffering from olive pollinosis [2]. Oth- ers proteins like Ole e 4, Ole e 9, Ole e 10, and Ole e 11 have been also reported as major allergens [1–4]. In addition, Ole e 6 and Ole e 7 allergens reach high values of clinical incidence [1, 2] and Ole e 2, Ole e 3, and Ole e 7 are involved in cross- reactivities, being classified as panallergens. Some of these proteins exhibit a significant polymorphism in 1-D and 2-D polyacrylamide gels, shown as a pattern of multiple bands or spots, respectively [4,5]. This variability is due to the existence of several protein forms of the allergen as a consequence of amino acid substitutions, PTMs (e.g. glycosylation), and/or Correspondence: Dr. Juan de Dios Alch ´ e, Department of Biochem- istry, Cell and Molecular Biology of Plants Estaci ´ on Experimental del Zaid´ ın (CSIC) Profesor Albareda 1, E-18008, Granada, Spain E-mail: juandedios.alche@eez.csic.es Fax: +34-95-812-9600 Abbreviations: Ab, antibody; SOD, superoxide dismutase the presence of multimeric forms [4,5]. Moreover, some aller- gens show conspicuous quantitative differences, depending on the genetic origin (i.e. cultivar) of pollen [6, 7]. Currently, this molecular variability is underrepresented in the com- mercial protein extracts of olive pollen used for diagnosis and treatment of olive pollen allergy. The relative abundance of individual protein forms of an allergen with distinct IgE- reactivity might affect the total allergenic potency of the ex- tract. The standardization of commercial olive pollen extracts is of great importance in order to warrant efficiency and safety in the diagnosis and immunotherapy procedures. Therefore, it is important to ensure that the allergenic variability in stan- dardized protein extracts resembles as much as possible that observed in the natural sources. Current procedures for preparation of protein extracts frequently determine the IgE response of a population, rep- resented by a pool of sera from allergic patients. Analysis of patient’s reactivity is performed in vivo by SPT (skin prick test), as well as in vitro by competitive assays like RAST (radioallergosorbent test) and ELISA inhibition tests among others. These procedures do not routinely discriminate indi- vidual allergens in the case of olive, and hence do not explain their individual contribution to reactivity. Therefore, further ∗ These authors have contributed equally to this work. ∗∗ Current address: Proteomic Research Service, Hospital Universitario San Cecilio, Avda. Dr. Ol ´ oriz 16, E-18012, Granada, Spain Colour Online: See the article online to view Figs. 1 and 4 in colour. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com