甘草酸单克隆抗体的制备与鉴定

目的: 制备特异性的甘草酸(GA)单克隆抗体。 方法: 用合成的甘草酸-牛血清白蛋白(GA-BSA)人工抗原免疫BALB/c小鼠;待血清检测阳性后,取其脾细胞与小鼠骨髓瘤细胞SP2/0按10∶1的比例融合;用间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;用阳性细胞株诱导制备腹水抗体;用辛酸硫酸铵法纯化抗体,并进行交叉反应检测。 结果: 获得了稳定分泌抗甘草酸的单克隆抗体杂交瘤细胞株GA-Mab-DF5,腹水抗体纯化后纯度达98%,回收率达80%。线性检测范围为4~64 μg·mL^-1,R²=0.9986,并且与BSA、明胶、胆酸、去氧胆酸、芍药苷、黄芩苷等无交叉反应。 结论: 成功获得能稳定分泌甘草酸单克隆抗体的杂交瘤细胞株,灵敏度、特异性均较高,为样品中甘草酸的快速微量检测及免疫亲和色谱柱制备奠定了基础。

Objective: To prepare a specific monoclonal antibody against glycyrrhizic acid(GA). Methods: BALB/c mice were immunized with the prepared GA-BSA conjugate.When the serum detection was positive,spleen cells from the mice were isolated and fused with SP2/0 myeloma cells at a ratio of 10∶1.Monoclonal hybridoma cells were screened by indirect ELISA and limited dilution.The ascites antibodies were induced and prepared by the positive cell line,and then were purified by caprylic acid/ammonium sulfate precipitation and tested by cross reaction. Results: The monoclonal antibody hybridoma cell line GA-Mab-DF5 steadily secreting glycyrrhizic acid antibodies was obtained,the purity of ascites antibodies was up to 98%,and the recovery rate was 80%.The linear range was 4 μg·mL^-1 to 64 μg·mL^-1(R²=0.9986)and no cross-reactivity was detected with BSA,gelatin,cholic acid,deoxycholic acid,paeoniflorin,baicalin and so on. Conclusion: The anti-GA antibody-secreting hybridomas are obtained with high sensitivity and specificity,which lays a foundation for the trace detection and immunoaffinity column preparation of glycyrrhizic acid in samples.