Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs

Abstract

A single-chain variable fragment antibody (scFv) against ginsenoside Re (G-Re) was constructed and applied to an enzyme-linked immunosorbent assay (ELISA) for determining the total concentration of ginsenosides in various ginsengs. The variable heavy and light chain genes were cloned directly from the cDNA of the 4G10 hybridoma cell line and assembled by means of splicing by overlapping extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (Gly(4)Ser)(3) between the variable heavy chain and light chain domains. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The recombinant scFv against G-Re (GRe-scFv) was expressed as a chimera protein containing the His6-tag at its N-termini, purified by immobilized metal ion affinity chromatography (IMAC), and refolded by a stepwise dialysis method. The yield of GRe-scFv after purification was 1.7 mg per liter of culture medium. Characterization of GRe-scFv revealed that it retained the characteristics of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10) which has wide cross-reactivity with 20(S)-protopanaxadiol- and 20(S)-protopanaxatriol-type ginsenosides. The detectable range for G-Re in ELISA using scFv antibody was 0.02-10 µg/ml. Based on validation analysis, the use of GRe-scFv in ELISA is a precise, accurate, and sensitive method. In light of the time-consuming and labor-intensive procedures for the preparation of MAb, speedy bacterial expression of GRe-scFv is a powerful alternative tool for producing MAb to use in ELISA for quantitative analysis of total ginsenoside concentrations.

Recent Methodology in Ginseng Analysis

“…Pongkitwitoon et al . [153] and Sritularak et al . [154] used a monoclonal antibody against specific ginsenosides for the ELISA-based determination of ginsenoside Rb 1 , Rg 1 , and Re in American ginseng berries and flowers [153,154].…”

Ginsenoside Re Promotes Nerve Regeneration by Facilitating the Proliferation, Differentiation and Migration of Schwann Cells via the ERK- and JNK-Dependent Pathway in Rat Model of Sciatic Nerve Crush Injury

“…Ginseng belongs to the genus Panax, and ginsenosides are the major active ingredients in ginseng (Pongkitwitoon et al 2011). More than 40 species of ginsenoside monomers are found and among them Re is a chief one (Peng et al 2012).…”

Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

“…However, in the case of N-format fluobody, the signal was undetectable due to its low fluorescence intensity as well as in indirect FLISA, even though competitive inhibitory activity was detected in the ELISA system. More interestingly, both limits of detection (LOD) for PL and G-Re determinations in FLISA system using the C-fluobody were found to be about 10-fold lower than that in the conventional ELISA using scFv (PL-scFv and GRe-scFv) [40,41] and their parental MAb (MAb 3A3 and MAb-4G10) [16,17], where the LOD for PL and G-Re showed 200 ng/mL and 100 ng/mL, respectively. It was estimated that the improvement of LOD comes from the highly sensitive fluorescence of AcGFP detected by the fluorescent microplate reader compared to that of the enzyme.…”

“…The developed ELISA using MAbs showed potential as an accurate and reliable assay for assessing the quality of the host plant. We subsequently constructed scFv antibody against PL (PL-scFv) and G-Re (GRe-scFv) using the cDNA of their hybridoma cell lines secreting MAb 3A3 and MAb-4G10, respectively, and expressed in E. coli, Sf9 insect cells, and silkworm (Bombyx mori) to obtain the probes for ELISA more efficiently [40][41][42][43]. However, an even simpler, speedy, and sensitive immunoassay is required to deal with a large number of plant samples serially.…”