Preparation of a highly specific single chain variable fragment antibody targeting miroestrol and its application in quality control of Pueraria candollei by enzyme‐linked immunosorbent assay

Abstract

Introduction Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products. Objectives To prepare a single‐chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv‐based enzyme‐linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products. Methods A gene encoding anti‐miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti‐miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv‐based ELISA method was validated for its sensitivity, specificity, accuracy and precision. Results Anti‐miroestrol scFv antibody was highly specific to miroestrol. The scFv‐based ELISA was applied to determine miroestrol in the range 0.06–7.81 μg/mL, with the limit of quantification of 0.06 μg/mL miroestrol. The accuracy of the assay was validated by its 95.08–103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody‐based ELISA. The relative standard deviation of the intra‐ and inter‐assay were less than 6.0%. Conclusion The developed scFv‐based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.

A novel chitosan–PEG hydrogel embedded with in situ silver nanoparticles of Clerodendrum glandulosum Lindl. extract: evaluation of its in vivo diabetic wound healing properties using an image-guided machine learning model

“…This activity of the CGH formulation can be explained by the presence of polyphenols and phenylethanoid glycosides in the formulated CG-AgNPs, which are highly anti-inflammatory in nature. 22,81,82 The in vivo diabetic wound healing study revealed that CGH nearly exhibited complete wound closure within 15 days compared to the BH and standard formulation, as evaluated through the developed machine learning model based on the provided datasets. Accordingly, a correlation was successfully established between the percentage of wound area and the elapsed number of days (Fig.…”

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen‐binding antibody produced via Escherichia coli

“…8 Consequently, with this E. coli strain, antibodies can be obtained in their active state, thereby making them suitable for use in immunoassays and other applications. E. coli SHuffle ® T7 is used to produce active scFv antibodies against small molecules, such as miroestrol 9 and ganoderic acid A, 10 which can be applied in ELISA procedures.…”

Protective effect of Tecomastans (L.) Juss.exKunth in CFA-induced arthritic rat model

“…In IL-1β, verbascoside exhibited a better docking score due to its hydrogen bond interactions with amino acids LYS103, VAL155, GLU153, GLN166, and ALA85 and electrostatic interactions with GLU165 and hydrophobic interactions with ALA83, ALA84, PRO40, ALA83, ALA85, and LYS103.In TNF-α, verbascoside demonstrated the highest docking score primarily due to its hydrogen bond interaction with TYR151 and hydrophobic interactions with TYR59 and LEU157.Likewise, verbascoside achieved a superior docking score against IL-6, which is attributed to its hydrogen bond interaction with GLU23 and hydrophobic interaction with LYS27.This aligns with punicalagin, a polyphenolic compound that has shown substantial binding a nities to identical protein targets via hydrogen bonding and hydrophobic interactions (Jghef et al 2023). Furthermore, verbascoside has been reported to exhibit potent anti-in ammatory activities by suppressing the expression of COX-2 (Pongkitwitoon et al 2024). A decrease in TNF-α, IL-1β, and IL-6 levels also has been observed in rats administered with verbascoside (Rossi et al 2023).…”