一种基于单克隆抗体的竞争性酶联免疫吸附测定法，用于测定中药及生物样品中的鲁斯可皂苷元。

A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples.

作者信息

Xu Yu, Liu Ji-Hua, Wang Jing, Zhang Jian, Yu Bo-Yang

机构信息

Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China.

出版信息

Chin J Nat Med. 2014 Oct;12(10):794-9. doi: 10.1016/S1875-5364(14)60121-7. Epub 2014 Oct 31.

DOI:10.1016/S1875-5364(14)60121-7

PMID:25443374

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine ruscogenin (RUS) by using the monoclonal antibody (McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin (15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng·mL(-1) and a detection limit (IC20) of 20.57 ng·mL(-1), was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.

摘要

开发了一种竞争性酶联免疫吸附测定法（ELISA），以使用单克隆抗体（McAb）来测定鲁斯可皂苷元（RUS）。从建立的杂交瘤细胞系分泌的抗RUS单克隆抗体被鉴定为IgG1同种型。该单克隆抗体对RUS表现出高特异性，与薯蓣皂苷元有非常轻微的交叉反应性（15.7%），对菝葜皂苷元、甘草酸二铵、齐墩果酸和三七皂苷R1无交叉反应性。所建立的ELISA的半数抑制浓度（IC50）值为157.55 ng·mL(-1)，检测限（IC20）为20.57 ng·mL(-1)，与高效液相色谱（HPLC）分析进行比较，在麦冬提取物中RUS的ELISA和HPLC-蒸发光散射检测（HPLC-ELSD）分析之间获得了良好的相关性。实验数据表明，ELISA方法相对于HPLC-ELSD具有更多优势，如检测限低、特异性高、背景低且无需样品预处理，更适合于中药中天然成分的测定以及用于药代动力学研究的生物样品的测定。