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

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 À1 with a limit of detection of 1.28 ng mL À1, 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.

A monoclonal antibody‐based indirect competitive enzyme‐linked immunosorbent assay to quantify swertiamarin and related compounds in Swertia japonica Makino

“…We isolated amaroswerin from S. japonica in previous work. 24 BSA (≥ 97%), albumin from chicken egg white (OVA, 98%), hybri-max polyethylene glycol (PEG), and CDI were purchased from Sigma-Aldrich (St Louis, MO, USA). Complete and incomplete Freund's adjuvants were obtained from Difco (Detroit, MI, USA).…”

“…The SM-cBSA conjugate was used as an immunogenic agent to immunise the mice. [24][25][26] The SM-cBSA conjugate was administered intraperitoneally to the BALB/c mice every 2 weeks. For the initial immunisation, the immunogenicity of the SM-cBSA conjugate (50 μg) was enhanced by adding Freund's complete adjuvant to the formulation.…”

Lateral flow immunoassay for small-molecules detection in phytoproducts: a review

“…The compounds with carboxylic acid and hydroxyl functional groups are usually conjugated by a carbodiimidemediated reaction [33,34,47,[95][96][97][98][99][100][101][102], whereas compounds with active hydrogen, e.g., α-picolines, ketones, esters, and acetylenes, can be conjugated to an amine or amide group of the carrier by a Mannich reaction [103,104]. Furthermore, structure modifications of the parent compounds to add the reactive functional group to the molecule can be conducted when necessary.…”