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 (Gly4Ser)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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Acknowledgments
This work was funded by a Grant in Aid from the Japan Society for the Promotion of Science Asian CORE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The research in this paper was also supported, in part, by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.
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B. Pongkitwitoon and S. Sakamoto contributed equally to this work.
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Pongkitwitoon, B., Sakamoto, S., Morinaga, O. et al. Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs. J Nat Med 65, 24–30 (2011). https://doi.org/10.1007/s11418-010-0446-1
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DOI: https://doi.org/10.1007/s11418-010-0446-1