Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase

We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimylistoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an α-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.