Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay.

Seiichi Sakamoto,Satoshi Morimoto,Shinji Wada,Hiroyuki Tanaka

doi:10.1039/c8ra02922a

Seiichi Sakamoto, Satoshi Morimoto + Show 2 more

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https://doi.org/10.1039/c8ra02922a

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Abstract

Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.

Highlights

Amarogentin [AG; Fig. 1(A)] is a naturally occurring secoiridoid glycoside produced mainly in plants belonging to the genera Swertia and Gentiana, both belonging to the family Gentianaceae
We focused on the quantitative analysis of plant secondary metabolites and developed various immunoassays, including enzyme-linked immunosorbent assays (ELISAs), uorescence-linked immunosorbent assays, and immunochromatographic strip assays
40 years ago, the appropriate hapten numbers had already been investigated by Erlanger for the production of antihapten antibodies using bovine serum albumin (BSA) conjugates and he concluded that between 8 and 25 molecules were optimal.[19]

Summary

Introduction

Amarogentin [AG; Fig. 1(A)] is a naturally occurring secoiridoid glycoside produced mainly in plants belonging to the genera Swertia and Gentiana, both belonging to the family Gentianaceae. We focused on the quantitative analysis of plant secondary metabolites and developed various immunoassays, including enzyme-linked immunosorbent assays (ELISAs), uorescence-linked immunosorbent assays, and immunochromatographic strip assays. These immunoassays exhibited several advantages over traditional analytical methods, such as HPLC, in terms of simplicity, selectivity, and sensitivity because the principle is based on a speci c antigen– antibody reaction.[15]

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