Generation and characterization of mouse monoclonal antibodies specific for N-linked neutral oligosaccharides of glycoproteins.

We generated four monoclonal antibodies (MAbs) specific for asparagine-linked neutral oligosaccharides of glycoproteins by immunizing mice with neoglycolipids, which were derived from glycoproteins by conjugation to phosphatidylethanolamine dipalmitoyl. The binding specificity of these MAbs was determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. The four MAbs designated OMB3, OMB4, OMR5, and OMR6 reacted strongly with the neoglycolipids, Gal beta1-4GlcNAc beta1-2Man alpha1-6(Gal beta1-4GlcNAc beta1-2Man alpha1-3)Man beta1-4GlcNAc-PD, GlcNAc beta1-2Man alpha1-6(GlcNAc beta1-2Man alpha1-3)(GlcNAc beta1-4)Man beta1-4GlcNAc beta1-4GlcNAc-PD, Man alpha1-6Man beta1-4GlcNAc beta1-4(Fuc alpha1-6)GlcNAc-PD, and Man alpha1-3Man beta1-4GlcNAc-PD, respectively, that were used as immunogens. All of these MAbs exhibited a high binding specificity. The epitopes of the MAbs OMB3 and OMB4 were suggested to be nonreducing terminal trisaccharides, Gal beta1-4GlcNAc beta1-2Man-, and nonreducing beta-GlcNAc residues, respectively. MAbs OMR5 and OMR6 showed a highly restricted binding specificity, reacting only with the immunizing neoglycolipids. Subsequently, MAbs OMB3 and OMB4 were shown to react strongly with asialo-alpha1-acid-glycoprotein and asialo-agalacto-alpha1-acid-glycoprotein, respectively, by Western blotting. Furthermore, it was shown that these MAbs reacted specifically with the epitope on Chinese hamster ovary cells by an immunofluorescence technique. MAb OMB4 was also shown to detect the accumulated oligosaccharides with nonreducing terminal beta-GlcNAc residues as granular inclusions in the cultured fibroblasts from a classical Sandhoff disease patient.