红景天苷单克隆抗体的制备及间接竞争酶联免疫方法的建立  

Preparation of Monoclonal Antibodies for Development of Indirect Competitive ELISA for Salidroside Detection

作　　者：王伟 胡立新 伏光华 张海丽[2] WANG Wei;HU Li-xin;FU Guang-hua;ZHANG Hai-li(Linyi Institute for Food and Drug Control,Linyi, Shandong276000, China;Heze University, Heze, Shandong 276000, China)

机构地区：[1]临沂市检验检测中心,山东临沂276000 [2]菏泽学院,山东菏泽274000

出　　处：《福建农业学报》2019年第5期595-599,共5页Fujian Journal of Agricultural Sciences

基　　金：山东省自然科学基金青年基金项目(ZR2014CQ057);菏泽学院博士基金项目(XY14B001)

摘　　要：【目的】制备红景天苷单克隆抗体,建立红景天苷间接竞争酶联免疫方法,实现经济、高效、简便快速批量检测红景天药材及其制品中的红景天苷含量,为其商品化检测试剂盒开发奠定基础。【方法】用已经成功偶联的红景天苷-BSA作为免疫原,免疫6~8周龄Balb/c小鼠,ELISA检测血清效价,取效价最高的小鼠脾细胞与骨髓瘤细胞进行融合,经有限稀释法筛选、克隆后获得能够稳定产生红景天苷单克隆抗体的细胞株,体内诱生腹水制备大量红景天苷单克隆抗体。利用该抗体,建立红景天苷间接竞争酶联免疫分析方法并进行方法学确认,包括日内精密度、日间精密度、回收率等指标。【结果】建立的间接竞争酶联免疫吸附分析方法的线性范围为4.07~598.45ng·mL-1,检测限IC10为1.77ng·mL-1,半数抑制率IC50为49.33ng·mL-1,日内精密度和日间精密度均小于4.5%,加标样品的平均回收率为94.3%,红景天苷含量检测结果与高效液相色谱法一致。【结论】本研究成功制备了红景天苷单克隆抗体,建立了间接竞争酶联免疫分析方法,能够用于为红景天苷含量的快速检测提供依据。【Objective】To prepare monoclonal antibody of salidroside for the development of an indirect competitive enzyme linked immunosorbent assay (ic-ELISA) methodology to accurately and rapidly detect salidroside in medicinal materials.【Method】 Balb/c mice of 6-8 weeks old were immunized with the prepared salidroside-BSA.Specificity of the anti-serum was determined by ELISA.Spleen cells from the mice with positive result were fused with SP2/0 myeloma cells.Monoclonal hybridoma cells were screened by indirect ELISA with a limited dilution.Ascitic antibodies were induced and prepared from the positive cell line.Specific antibody against salidroside was used to establish ic-ELISA detection method that was verified by its specificity, precision and recovery rate on a known standard.【Result】The sensitivity of ic-ELISA was 49.33 ng·mL -1 with a liner range of 4.07-598.45 ng·mL -1 .The detection limit of the method was1.77 ng·mL -1 .The newly developed method detected salidroside in spiked samples with a recovery rate of 94.3% within the designed range.The relative standard deviation of the measurements was less than 4.5% on both intra- and inter-day assays.The determination by ic-ELISA agreed with that obtained by HPLC.【Conclusion】 The newly established ic-ELISA method based on the salidroside monoclonal antibody was considered appropriate for the detection.

关 键 词：红景天苷 单克隆抗体 间接竞争酶联免疫法 

分 类 号：S853.72[农业科学—临床兽医学]