甘草酸单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against glycyrrhizic acid

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4～64μg.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/ e mice were immunized with the prepared GA - BSA conjugate. When the serum detection was positive, spleen cellsfrom 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 pos-itive 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 antibod-ies was obtained,the purity of ascites antibodies was up to 98% ,and the recovery rate was 80%. The linear range was 4 4μg·mL^-1 to 64 4μg·mL^-1 （ R^2 = 0. 9986） and no cross - reactivity was detected with BSA, gelatin, cholicacid, 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 immunoaffinitycolumn preparation of glycyrrhizic acid in samples.