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

Abstract

An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article

Development of a Monoclonal Antibody-Based Immunochemical Assay for Liquiritin and Its Application to the Quality Control of Licorice Products

“…Monoclonal antibody (mAb)-based immunochemical assays are inexpensive, fast, highly specific, sensitive, and promising methods for the quantitative and/or qualitative analysis of natural bioactive compounds. 20 Thus, immunoassays are a highthroughput screening method for a large number of samples. In a previous study, mAbs were prepared against glycyrrhizin with a fixed analytical system using an antiglycyrrhizin mAb.…”

Development of Double Eastern Blotting for Major Licorice Components, Glycyrrhizin and Liquiritin for Chemical Quality Control of Licorice Using anti-Glycyrrhizin and anti-Liquiritin Monoclonal Antibodies

“…Conversely, liquiritin (Liq; Figure B), which is one of the main flavonoids in licorice has functional bioactivities including neuroprotective effects, antidepressant-like effects, and growth suppression of pathogenic intestinal bacteria. − However, the component profiles of wild licorice vary depending on the growing area, cultivating environment, and species type ( Glycyrrhiza uralensis , G. glabra , or G. inflata ). , Thus, effective quality control of licorice is necessary to survey the components profile of licorice and it might be implied that Liq is one of the key constituent in licorice as well as GL. Although several analytical methods mainly based on HPLC have been employed to evaluate the quality of licorice and its products, including confectioneries, liqueur, and food supplements, these current methods need complicated pretreatment, large amounts of organic solvent for preparing sample extracts and mobile phases, and expensive separation columns. − In addition, since HPTLC has been developed for quantitative and/or qualitative analysis of GL for marketed licorice roots and its products, the specificity is unsuitable for detecting target compounds in crude sample extract. , In comparison to these techniques, an immunoassay using a monoclonal antibody (mAb) could be more convenient, economic, specific, and rapid for quantitative and/or qualitative analysis of numerous samples. − …”

Development of a highly sensitive and specific monoclonal antibody based enzyme‐linked immunosorbent assay for the detection of a new β‐agonist, phenylethanolamine A, in food samples

“…Currently, the most useful immunoassays are enzyme‐linked immunosorbent assays (ELISAs). The significant advantages of ELISAs over the traditional instrumental methods are that they are inexpensive, fast, highly specific and sensitive, and enable quantitative and/or qualitative analysis of different compounds . ELISAs have been widely used for the determination of both large and small analytes such as proteins, microorganisms, antibiotics, hormones, pesticides, halogenated and aromatic pollutants, and even heavy metals in the biological, medical, agricultural, and environmental area .…”