Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2015-03, Vol.985, p.197-205
• Main Authors: Qu, Huihua, Wang, Yan, Shan, Wenchao, Zhang, Yue, Feng, Huibin, Sai, Jiayang, Wang, Qingguo, Zhao, Yan
• Format: Article
• Language: English
• Subjects: 20(R,S)-ginsenoside-Rg2; Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay - methods; Ginsenoside-Rh1; Ginsenosides - analysis; Ginsenosides - chemistry; Ginsenosides - isolation & purification; Immunoaffinity chromatography; Immunochromatography - methods; Indirect competitive enzyme-linked immunosorbent assay; Isomerism; Limit of Detection; Linear Models; Monoclonal antibody; Reproducibility of Results
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2015.01.037

•Hybridomas producing anti-ginsenoside-Rh1 MAbs were generated.•The anti-G-Rh1 MAbs were used to create icELISAs for identification of 20(S)-Rh1, Rg2.•The IAC provides a new approach and potential method for resolving epimers. 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.