Identification of Carbohydrate Structures of Glycoproteins by a Novel Method Utilizing a Lectin-Elisa

Abstract

The glycosylation of proteins has a strong influence on their biological function. In case of IgG antibodies changes in glycosylation pattern may lead to dramatic dysfunctions of the antibody dependent immune response of humans against infections and tumors. In contrast to earlier performed lectin-blotting techniques an ELISA assays were performed to analyse carbohydrate structures of native antibody molecules. These tests based on lectins were performed in microtiter plates. To test the specificity of the lectins to their corresponding carbohydrates a inhibition test with mono-and disaccharides was used. Using these techniques a polyreactive monoclonal human IgG antibody (CBGA1), its Fc-and Fab-fragment were analyzed for their glycosylation pattern. The Fab fragment contains carbohydrate structures of both the N- and Oglycosylation types. Different glycosylation patterns were detected at the Fab-and Fcfragments of the CBGA1 antibody. Most of the glycan structures in the Fab-fragment were found to be sialylated and fucosylated in contrast to the Fc-fragment which expressed large amounts of terminal galactose. In addition to the typical complex biantennary glycan structures at all fragments terminal mannose, which represents hybrid and/or high mannose structures, were detected.