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Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix

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Abstract

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95–125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph–mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

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Funding

This study was funded by the Japan Agency for Medical Research and Development AMED (Grant No. JP21mk0101200).

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Author notes

  1. Akihiro Ochi and Shunsuke Fujii have contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan

    Akihiro Ochi, Poomraphie Nuntawong, Satoshi Morimoto & Seiichi Sakamoto

  2. Faculty of Health Management, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, 859-3298, Japan

    Shunsuke Fujii

  3. Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, 859-3298, Japan

    Tomoe Ohta & Takuhiro Uto

  4. Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, 1-1-1 Daigaku-Dori, Yamaguchi, 756-0884, Japan

    Hiroyuki Tanaka

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  1. Akihiro Ochi
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Contributions

Seiichi Sakamoto and Hiroyuki Tanaka made substantial contributions to the conception and design of the study and supervised the research, along with Satoshi Morimoto and Takuhiro Uto. Akihiro Ochi, Shunsuke Fujii, and Tomoe Ohata conducted the experiments. Akihiro Ochi and Shunsuke Fujii conducted data analysis, wrote the first draft, and made equal contributions to this work (co-first authors). Seiichi Sakamoto and Poomraphie Nuntawong revised the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Correspondence to Seiichi Sakamoto.

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Ochi, A., Fujii, S., Ohta, T. et al. Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix. J Nat Med 78, 160–168 (2024). https://doi.org/10.1007/s11418-023-01753-3

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