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

Benyakan Pongkitwitoon, Seiichi Sakamoto, Osamu Morinaga, Thaweesak Juengwatanatrakul, Yukihiro Shoyama, Hiroyuki Tanaka, Satoshi Morimoto

研究成果: ジャーナルへの寄稿記事

10 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)24-30
ページ数7
ジャーナルJournal of Natural Medicines
65
発行部数1
DOI
出版物ステータス出版済み - 1 1 2011

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Ginsenosides
Single-Chain Antibodies
Immunosorbents
Panax
Assays
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
His-His-His-His-His-His
Enzymes
Genes
Affinity chromatography
Light
Ion chromatography
Dialysis
Hybridomas
Affinity Chromatography
Escherichia coli
Purification
Metal ions
Culture Media

All Science Journal Classification (ASJC) codes

  • Pharmaceutical Science
  • Drug Discovery
  • Complementary and alternative medicine
  • Organic Chemistry

これを引用

Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs. / Pongkitwitoon, Benyakan; Sakamoto, Seiichi; Morinaga, Osamu; Juengwatanatrakul, Thaweesak; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi.

:: Journal of Natural Medicines, 巻 65, 番号 1, 01.01.2011, p. 24-30.

研究成果: ジャーナルへの寄稿記事

Pongkitwitoon, Benyakan ; Sakamoto, Seiichi ; Morinaga, Osamu ; Juengwatanatrakul, Thaweesak ; Shoyama, Yukihiro ; Tanaka, Hiroyuki ; Morimoto, Satoshi. / Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs. :: Journal of Natural Medicines. 2011 ; 巻 65, 番号 1. pp. 24-30.
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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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AU - Morinaga, Osamu

AU - Juengwatanatrakul, Thaweesak

AU - Shoyama, Yukihiro

AU - Tanaka, Hiroyuki

AU - Morimoto, Satoshi

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