摘要
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.
出处
《药物分析杂志》
CAS
CSCD
北大核心
2013年第5期770-774,共5页
Chinese Journal of Pharmaceutical Analysis
基金
国家自然科学基金(30973709
81274043)
北京市重点实验室2011阶梯计划(Z111107055311080)
北京中医药大学创新团队
关键词
甘草酸
单克隆抗体
制备
鉴定
杂交瘤细胞株
免疫亲和色谱柱
酶联免疫吸附法
glycyrrhizic acid
monoclonal antibody
preparation
identification
hybridoma cell lines
immune affinitychromatography column
ELISA