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Planta Med 2016; 82(05): 432-439
DOI: 10.1055/s-0035-1568182
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody

Jiayang Sai
1   The Third Affiliated Hospital, Beijing University of Chinese Medicine, Beijing, China
,
Yan Zhao
2   School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China
,
Wenchao Shan
3   School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China
,
Baoping Qu
2   School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China
,
Yue Zhang
2   School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China
,
Jinjun Cheng
3   School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China
,
Huihua Qu
4   Center of Scientific Experiment, Beijing University of Chinese Medicine, Beijing, China
,
Qingguo Wang
2   School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China
› Author Affiliations
Further Information

Publication History

received 10 June 2015
revised 14 November 2015

accepted 22 November 2015

Publication Date:
29 January 2016 (online)

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Abstract

This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL−1. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00 %. The recovery rates ranged from 92.36 % to 101.00 %, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d.

Key words

Bupleurum - Apiaceae - Bupleuri radix - saikosaponin d - monoclonal antibody - ELISA - immunoaffinity column chromatography
 

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