| Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs. | |
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MedLine Citation:
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PMID: 20706797 Owner: NLM Status: In-Data-Review |
Abstract/OtherAbstract:
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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. |
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Authors:
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Benyakan Pongkitwitoon; Seiichi Sakamoto; Osamu Morinaga; Thaweesak Juengwatanatrakul; Yukihiro Shoyama; Hiroyuki Tanaka; Satoshi Morimoto |
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Publication Detail:
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Type: Journal Article Date: 2010-08-14 |
Journal Detail:
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Title: Journal of natural medicines Volume: 65 ISSN: 1861-0293 ISO Abbreviation: J Nat Med Publication Date: 2011 Jan |
Date Detail:
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Created Date: 2010-12-14 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101518405 Medline TA: J Nat Med Country: Japan |
Other Details:
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Languages: eng Pagination: 24-30 Citation Subset: IM |
Affiliation:
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Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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