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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 GMI lactone, showed restricted specificity, detecting only the GMI lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GMI) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD Ia 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 NeuAcα2 → 3Gal‐ sequence (GD Ia, GM3, GM Ib, GT Ib, and IV3Neu´‐nLC4Cer), but not with those having a NeuAcα2 → 8NeuAcα2 → 3Gal‐ sequence. On the other hand, MAb AMR 19 reacted with ganglioside lactones having a NeuAcα2 → 8NeuAcα2 → 3Gal‐ sequence (GD3, O‐Ac‐GD3, GD2, GD Ib, GT Ib, GQ Ib and GT Ia), but not with those having a NeuAcα2 →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.