PRODUCTION OF MONOCLONAL-ANTIBODIES SPECIFIC FOR GANGLIOSIDE GD3

Four kinds of anti-G(D3) monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside G(D3) inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside G(D3) variants [G(D3)(NeuAc-NeuAc), G(D3)(NeuAc-NeuGc), G(D3)(NeuGc-NeuAc), and G(D3)(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with G(D3)(NeuAc-NeuAc) and G(D3)(NeuAc-NeuGc) but not with G(D3)(NeuGc-NeuAc) or G(D3)(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside G(D3) [G(D3)(NeuAc-NeuAc) and G(D3)(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal-beta-1-3GalNAc, NeuAc-alpha-2-3Gal-beta-1-3GalNAc, or NeuAc-alpha-2-8NeuAc-alpha-2-3Gal-beta-1-3GalNAc residue to the Gal moiety of ganglioside G(D3) (G(D2), G(D1b), G(T1b), or G(Q1b), respectively), ganglioside G(T1a) having the same terminal NeuAc-alpha-2-8NeuAc-alpha-2-3Gal residue as ganglioside G(D3), other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha-2-8Sia-alpha-2-3Gal-beta-1-4Glc residue of ganglioside G(D3).