A sensitive and specific indirect competitive enzyme‑linked immunosorbent assay for the detection of icariin

Cheng,Jinjun; Zhang,Yue; Liu,Shuchen; Qu,Baoping; Zhang,Meiling; Liu,Xiaoman; Zhao,Yan; Qu,Huihua

doi:10.3892/mmr.2016.5975

A sensitive and specific indirect competitive enzyme‑linked immunosorbent assay for the detection of icariin

Authors:
- Jinjun Cheng
- Yue Zhang
- Shuchen Liu
- Baoping Qu
- Meiling Zhang
- Xiaoman Liu
- Yan Zhao
- Huihua Qu
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Affiliations: Department of Basic Traditional Chinese Medicine Clinical Science, School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100029, P.R. China, Department of Pharmaceutics of Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, P.R. China, Department of Chemistry of Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, P.R. China, Department of Molecular Biology, Beijing Institute of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, P.R. China
Published online on: November 30, 2016 https://doi.org/10.3892/mmr.2016.5975
Pages: 411-416

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Abstract

In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridoma‑secreting MAb against icariin was produced by fusing splenocytes immunized with an ICA‑bovine serum albumin conjugate with a hypoxanthine‑aminopterin‑thymidine‑sensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no cross‑reactivity against the majority of structurally‑related chemicals. Subsequently, an indirect competitive enzyme‑linked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 10‑1,000 ng/ml of ICA (R2=0.9828) was detected. Intra‑ and inter‑assay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95‑115% was obtained, with an RSD of <10%. In addition, the levels of ICA in traditional Chinese herbal prescriptions were determined, and correlation between the ELISA and high‑performance liquid chromatography analyses of total ICA was obtained. These results demonstrated that a reliable ELISA method had been successfully developed to determine ICA in traditional Chinese herbs and may contribute to further clinical investigations.

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