Kawashima I, Kotani M, Ozawa H, Suzuki M, Tai T
Department of Tumor Immunology, Tokyo Metropolitan Institute of Medical Science, Japan.
Int J Cancer. 1994 Jul 15;58(2):263-8. doi: 10.1002/ijc.2910580220.
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.
我们通过用吸附于明尼苏达沙门氏菌的纯化内酯免疫C3H/HeN小鼠，随后与小鼠骨髓瘤细胞融合，产生了3种对神经节苷脂内酯具有特异性的鼠单克隆抗体（MAb）。使用包括神经节苷脂内酯在内的多种糖脂，通过酶联免疫吸附测定（ELISA）以及在薄层色谱上进行免疫染色，得以阐明这些单克隆抗体所识别的精确结构。用GM1内酯产生的单克隆抗体AMR38显示出受限的特异性，仅检测用于免疫的GM1内酯。所测试的其他神经节苷脂内酯、完整神经节苷脂（包括GM1）或中性糖脂均未被识别。相比之下，分别用GD1a内酯和GD3内酯产生的单克隆抗体AMR40和AMR19显示出更广泛的特异性，识别多种神经节苷脂内酯。然而，精确的表位是不同的。单克隆抗体AMR40与具有外部NeuAcα2→3Gal序列（GD1a、GM3、GM1b、GT1b和IV3NeuAcα - nLc4Cer）的神经节苷脂内酯强烈反应，但不与具有NeuAcα2→8NeuAcα2→3Gal序列的神经节苷脂内酯反应。另一方面，单克隆抗体AMR19与具有NeuAcα2→8NeuAcα2→3Gal序列（GD3、O - Ac - GD3、GD2、GDlb、GTlb、GQlb和GTla）的神经节苷脂内酯反应，但不与具有NeuAcα2→3Gal序列的神经节苷脂内酯反应。所测试的完整神经节苷脂或中性糖脂均未被这些单克隆抗体识别。我们还通过免疫荧光技术确定了在无胸腺裸鼠中生长的人黑色素瘤细胞上神经节苷脂内酯的表达。
