Generation of monoclonal antibodies specific for ganglioside lactones: evidence of the expression of lactone on human melanoma cells.
We generated 3 murine monoclonal antibodies (MAbs) specific for ganglioside lactones by immunizing C3H/HeN mice with purified lactones adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. The use of a wide variety of glycolipids, including ganglioside lactones, enabled the precise structures recognized by these MAbs to be elucidated through an ELISA and by immunostaining on thin-layer chromatography. MAb AMR38, which was generated with GM1 lactone, showed restricted specificity, detecting only the GM1 lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GM1) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD1a lactone and GD3 lactone, respectively, showed broader specificities, recognizing several ganglioside lactones. However, the precise epitopes were different. MAb AMR40 reacted intensely with ganglioside lactones having an external NeuAc alpha 2-->3Gal-sequence (GD1a, GM3, GM1b, GT1b, and IV3NeuAc alpha-nLc4Cer), but not with those having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence. On the other hand, MAb AMR19 reacted with ganglioside lactones having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence (GD3, O-Ac-GD3, GD2, GDlb, GTlb, GQlb and GTla), but not with those having a NeuAc alpha 2-->3Gal- sequence. None of the intact gangliosides or neutral glycolipids tested were recognized by the MAbs. We also determined the expression of ganglioside lactones on human melanoma cells grown in athymic nude mice by means of an immunofluorescence technique.
Kawashima
I
I
Department of Tumor Immunology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kotani
M
M
Ozawa
H
H
Suzuki
M
M
Tai
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis*; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D002417:Cattle; D002855:Chromatography, Thin Layer; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005732:Gangliosides / Q000276:immunology*; D006017:Glycolipids / Q000032:analysis; D000882:Haplorhini; D006801:Humans; D007114:Immunization; D007150:Immunohistochemistry; D007783:Lactones / Q000276:immunology*; D008545:Melanoma / Q000737:chemistry*; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D011784:Quail; D012756:Sheep; D013552:Swine
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D006017:Glycolipids; D007783:Lactones