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 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.
Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas-CSIC, Granada, Spain.
Sonia
S
Morales
Antonio Jesús
AJ
Castro
Jose Carlos
JC
Jimenez-Lopez
Fernando
F
Florido
María Isabel
MI
Rodríguez-García
Juan
J
de Dios Alché
D000485:Allergens / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D051379:Mice; D031658:Olea / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011058:Pollen / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits; D006255:Rhinitis, Allergic, Seasonal / Q000097:blood; D012698:Serologic Tests / Q000379:methods / Q000592:standards
D000485:Allergens; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010940:Plant Proteins; D007073:Immunoglobulin E