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 neo-glycolipids, 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 beta 1-4GlcNAc beta 1- 2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3) Man beta 1-4GlcNAc-PD, GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3) (GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4GlcNAc-PD, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-PD, and Man alpha 1-3Man beta 1-4GlcNAc-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 beta 1-4GlcNAc beta 1-2Man-, 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-alpha(1) -acid-glycoprotein and asialo-agalacto-alpha(1)-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. (C) 1997 Academic Press.