Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides

Abstract

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGcα2→3Gal- terminal structures, such as GM3(NeuGc), IV 3NeuGcα-Gg 4Cer, IV 3NeuGcα-nLc 4Cer, V 3NeuGcα-Gb 5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGcα2→3Gal-sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAcα2→3Gal-sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGcα2→3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GDS(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGcα2→8NeuGcα2→3Gal-terminal sequences, such as GD3(NeuGc-NeuGc-),IV 3NeuGcα 2-Gg 4Cer,IV 3NeuGcα 2-nLc 4Cer, and V 3NeuGcα 2-Gb 5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGcα2→8NeuGcα2→3Gal-terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.