title: Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers.
abstract: In this work, hybridomas producing anti-ginsenoside-Rh1 monoclonal antibodies (MAbs) were generated. These MAbs were subsequently used to create indirect competitive enzyme-linked immunosorbent assays (icELISAs). A linear correlation was obtained for G-Rh1 concentrations in the range from 26 to 512ng/mL. The regression equation was y=1.979-0.201Log2(X) with a regression coefficient of 0.9898. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay). The recovery rates ranged from 93.16% to 108.43%. Testing with the icELISA demonstrated that the MAbs were specific for 20(S)-Rh1 and 20(S)-Rg2 with no cross-reactivity against 20(R)-Rh1 and 20(R)-Rg2. The immunoaffinity chromatography column (IAC) was constructed by covalently coupling monoclonal antibody (MAb) against G-Rh1 to CNBr-activated Sepharose 4B. When 20(R)-type-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-type-Rg2 ran through the column. The differences in adsorption between the 20(S) and 20(R) type ginsenosides bring a new approach or method to separate 20(S)-Rg2 and 20(R)-Rg2 by IAC. Our results indicate that the icELISA is a sensitive and efficient approach for the identification of epimers, and the application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving epimers.
authors.affiliation: Center of Scientific Experiment, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Qu
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Shan
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Huibin
authors.initials: H
authors.lastname: Feng
authors.affiliation: The third affiliated hospital, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Jiayang
authors.initials: J
authors.lastname: Sai
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Wang
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China. Electronic address: zhaoyandr@gmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D036145:Ginsenosides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D007536:Isomerism; D057230:Limit of Detection; D016014:Linear Models; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D036145:Ginsenosides; C026474:ginsenoside Rg2; C425564:ginsenoside Rh1
keywords: 20(R,S)-ginsenoside-Rg2; Ginsenoside-Rh1; Immunoaffinity chromatography; Indirect competitive enzyme-linked immunosorbent assay; Monoclonal antibody