A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens

Background Carbohydrate epitopes are capable of binding human IgE from alle rgic subjects and these epitopes play a role in the cross-reactivity betwee n allergens from unrelated sources. A monoclonal antibody (5E6), specific f or a carbohydrate epitope detectable on components of Cupressus arizonica p ollen extract, has been produced and characterized. Objective To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the p resence of such carbohydrate IgE determinant in extracts from 21 pollen spe cies belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. Methods IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fractio n were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase , adsorbed on the ELISA plates. The relationship between the monoclonal- an d the IgE-recognized epitopes was investigated by ELISA-competition experim ents. Analysis of the distribution of this carbohydrate epitope was perform ed by direct binding of the monoclonal antibody onto the various extracts. Results The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine ox idase. Polyamine oxidase shows only one N-glycosilation site whose carbohyd rate moiety seems to be composed of a branched chain of seven ordered sugar s, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xy lose-residues. This structure bears the epitope recognized by mAb 5E6. Huma n IgE from the IgG-depleted fraction were found capable of inhibiting the m onoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 rang ing from 0.110 to 2.060). Conclusion Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects a n epitope which is also recognized by IgE from allergic subjects. This char acterized reagent could be a useful tool for studying distribution of cross -reactive carbohydrate determinants in allergenic pollen extracts and their components.