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Analytical study of cypress pollen allergy: molecular and particular aspects
The cypress pollen represents a widespread and highly invasive allergenic source worldwide. In the context of this study, we focused on the identification and characterization of the main allergens implicated in the cypress pollen (CP) allergy using a proteomic approach. We have developed novel detergent-based pollen protein extraction protocols in order to solubilise both hydrophilic and hydrophobic proteins from CP. One- and two-dimensional-PAGE (2-DE) immunoblotting were used for the immunochemical characterization of CP proteins using sera from cypress allergic patients living in France and Italy (n= 120). The identification of allergens was carried out by in-gel excision and tandem mass spectrometry (MS/MS) analysis of 2-DE IgE binding spots. The biochemical characterization of novel allergens included determination of isoelectric points, molecular weights, the presence of bromelain-type CCD, concanavalin A–binding ability, heat stability and the degree of allergenicity under reducing and denaturing conditions. We have designed a new methodology based on double one-dimensional electrophoresis (D1-DE) for the IgE screening test of allergenic components overlapping in 1-D electrophoresis. D1-DE also provides high resolution for allergenic proteins in a narrow pI-range and represents a rapid alternative to 2-DE for the simultaneous one-step IgE screening of allergic patients. The IgE repertoire diversity of French and Italian patients was also analyzed by using a microarray system including 103 different allergens and correlated with the established IgE immunoblot profiles. Combinatorial peptide ligand libraries (CPLL) were used for capturing the low- and very low-abundance proteins. Different eluates from the CPLL have been resolved by two-dimensional mapping, blotted and confronted with sera from cypress allergic patients. Novel low-abundant allergens were identified by using MS/MS analysis.
The panel of preparative and analytical methods developed through this study revealed on the one hand, the diversity of the IgE responses among cypress pollen allergic patients and, on the other, the diversity of the allergens implicated in this pathology.