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目的制备雪卡毒素的单克隆抗体并对其生物学特性进行鉴定。方法以雪卡毒素类似物莫能菌素为半抗原,分别采用混合酸酐法将其与载体牛血清白蛋白(BSA)偶联合成免疫抗原M-BSA,碳二亚胺法与卵清白蛋白(OVA)偶联合成包被抗原M-OVA。以M-BSA免疫Balb/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合制备杂交瘤。以自制雪卡毒素提取物为竞争抗原,间接竞争ELISA法筛选稳定分泌雪卡毒素抗体的特异性细胞株。小鼠体内诱生腹水,辛酸硫酸铵沉淀法进行纯化。采用抗体亚型鉴定试剂盒鉴定所得抗体的亚型,间接竞争ELISA法鉴定抗体的特异性和交叉性。结果获得3株稳定分泌抗雪卡毒素抗体的杂交瘤细胞株1D5、2G11、3G11,其Ig亚型均属于IgG1亚型。交叉反应结果表明3株单克隆细胞产生的抗体除与莫能菌素有较高的交叉反应外,与其他雪卡毒素类似物均无明显交叉反应。结论成功筛选到了分泌雪卡毒素抗体的细胞株,为雪卡毒素免疫分析方法的进一步研究奠定基础。
Objective To prepare ciguatoxin monoclonal antibodies and to identify their biological characteristics. Methods Monoclonal antigens of Ciguatoxin analogues were used as hapten and conjugated with carrier bovine serum albumin (BSA) to synthesize M-BSA, carbodiimide method and ovalbumin OVA) to coat the antigen M-OVA. Balb / c mice were immunized with M-BSA and their spleen cells were fused with Sp2 / 0 myeloma cells to prepare hybridomas. The homemade ciguatoxin extract was used as competitive antigen, and indirect competitive ELISA was used to screen the specific cell line which can stably secrete ciguatoxin antibody. In vivo induced ascites in mice, octanoic acid ammonium sulfate precipitation method for purification. The subtype of the obtained antibody was identified by using the antibody subtype identification kit, and the specificity and the cross resistance of the antibody were identified by indirect competition ELISA. Results Three hybridoma cell lines, 1D5, 2G11 and 3G11, secreting anti-ciguatoxin antibodies were obtained. All of their Ig subtypes belong to IgG1 subtype. Cross-reaction results showed that the antibodies produced by the three monoclonal cells had no significant cross-reaction with other ciguatoxin analogs, except for the higher cross-reaction with monensin. Conclusion The cell lines secreting ciguatoxin antibody were successfully screened, which laid the foundation for the further study of ciguatoxin immunoassay.