Production of Monoclonal Antibodies Specific for Ganglioside G_D3

Abstract

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 lipopoly-saccharides. 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 Ga1NAc, Galβ1-3Ga1NAc, NeuAcα2-3Galβ1-3GalNAc, or NeuAcα2-8NeuAc α2-3Ga1β1-3GalNAc 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 NeuAcα2-8NeuAcα2-3Gal 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-α2-8Siaα2-3Galfβl-4Glc residue of ganglioside G_D3.