ABSTRACT
BACKGROUND: Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra. OBJECTIVE: Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort. METHODS: Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed. RESULTS: 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs). CONCLUSION: We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.
Subject(s)
Allergens/analysis , Hypersensitivity, Immediate/etiology , Plant Extracts/chemistry , Sambucus nigra , Allergens/genetics , Animals , Base Sequence , Cross Reactions , Flowers , Fruit , Humans , Hypersensitivity, Immediate/immunology , Immunoglobulin E/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , N-Glycosyl Hydrolases/genetics , Plant Extracts/immunology , Plant Proteins/genetics , Pollen , Ribosome Inactivating Proteins, Type 2 , Sambucus nigra/immunology , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
A simple, but highly efficient, method was developed for the purification of monospecific antibodies against the plant glycoprotein Sambucus nigra lectin related protein. In a first step, the antiserum is purified by affinity chromatography on a column with the immobilized antigen. To deplete the affinity-purified antiserum from aspecific cross-reacting antibodies directed against the glycan part of the glycoprotein, a second affinity chromatography on an unrelated plant glycoprotein, in casu the Robinia pseudoacacia agglutinin, is included.
