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A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens
Author(s) -
Morales Sonia,
Castro Antonio Jesús,
Jimenez-Lopez Jose Carlos,
Florido Fernando,
RodríguezGarcía María Isabel,
de Dios Alché Juan
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100667
Subject(s) - multiplex , polyclonal antibodies , pollen , allergen , blot , western blot , chemistry , microbiology and biotechnology , monoclonal antibody , antibody , chromatography , immunoassay , biology , biochemistry , allergy , botany , immunology , gene , genetics
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 W estern blotting method for the simultaneous detection of four olive pollen allergens ( O le e 1, O le e 2, O le e 5, and O le e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated F ab 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 I g E ‐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 I g E ‐reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the I g E ‐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.