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.
Highlights
Antibody-based analytical methods have recently received much attention due to high-binding affinity and specificity of antibodies to target molecules
To fuse scFv with AcGFP domains by Splicing by overlap extension PCR (SOE-PCR), they were primarily amplified by PCR from pET28a expression vector encoding PL-scFv and GRe-scFv gene and pAcGFP1-N1 vector encoding AcGFP gene
We have mainly focused on the preparation of monoclonal antibodies against natural products and the application in enzyme-linked immunosorbent assay (ELISA) for the serial determination of bioactive compounds in medicinal plants in order to select high quality strains after breeding [12]
Summary
Antibody-based analytical methods have recently received much attention due to high-binding affinity and specificity of antibodies to target molecules. Ginsenosides are major triterpenoid saponins mainly produced and isolated from the root of Panax ginseng (white ginseng) and its related species including P. japonicus (Japanese ginseng), P. quinquefolium (American ginseng), and P. notoginseng (Tienchi ginseng) Their demand has been dramatically increased worldwide as ingredients of dietary health supplements and additives in foods and beverages due to their pharmacological activities such as tonic, immunomodulatory, antimutagenic, and anti-aging activities [30,31]. To avoid the disadvantages of chemical conjugation, a fluorescent single domain antibody (fluobody) which is a fusion protein of a green fluorescent protein (GFP) and a scFv antibody has been genetically constructed and used alternatively In this genetically engineered format, the resultant protein is always expressed in a one-to-one ratio between the fluorochrome and scFv, which should enhance the accuracy of the quantitative analysis. The potential use of a fluobody in quantitative/qualitative analysis of bioactive natural products has been reviewed in this article
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