Bacterial expression of a single-chain variable fragment (scFv) antibody against ganoderic acid a: A cost-effective approach for quantitative analysis using the scfv-based enzyme-linked immunosorbent assay

Gorawit Yusakul, Poomraphie Nuntawong, Seiichi Sakamoto, Pahweenvaj Ratnatilaka Na Bhuket, Toshitaka Kohno, Nao Kikkawa, Pornchai Rojsitthisak, Kuniyoshi Shimizu, Hiroyuki Tanaka, Satoshi Morimoto

研究成果: ジャーナルへの寄稿記事

1 引用 (Scopus)

抄録

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle® strain. Purified anti-GAA scFv, which yielded 2.56mg from 1L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078–1.25µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1–101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.

元の言語英語
ページ(範囲)1767-1774
ページ数8
ジャーナルBiological and Pharmaceutical Bulletin
40
発行部数10
DOI
出版物ステータス出版済み - 1 1 2017

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Single-Chain Antibodies
Enzyme-Linked Immunosorbent Assay
Costs and Cost Analysis
Immunoassay
Ganoderma
Reishi
Escherichia coli
Phytochemicals
Antibody Formation
Culture Media
ganoderic acid
ganoderic acid A
Cytoplasm
Antibodies

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

これを引用

Bacterial expression of a single-chain variable fragment (scFv) antibody against ganoderic acid a : A cost-effective approach for quantitative analysis using the scfv-based enzyme-linked immunosorbent assay. / Yusakul, Gorawit; Nuntawong, Poomraphie; Sakamoto, Seiichi; Bhuket, Pahweenvaj Ratnatilaka Na; Kohno, Toshitaka; Kikkawa, Nao; Rojsitthisak, Pornchai; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi.

:: Biological and Pharmaceutical Bulletin, 巻 40, 番号 10, 01.01.2017, p. 1767-1774.

研究成果: ジャーナルへの寄稿記事

Yusakul, Gorawit ; Nuntawong, Poomraphie ; Sakamoto, Seiichi ; Bhuket, Pahweenvaj Ratnatilaka Na ; Kohno, Toshitaka ; Kikkawa, Nao ; Rojsitthisak, Pornchai ; Shimizu, Kuniyoshi ; Tanaka, Hiroyuki ; Morimoto, Satoshi. / Bacterial expression of a single-chain variable fragment (scFv) antibody against ganoderic acid a : A cost-effective approach for quantitative analysis using the scfv-based enzyme-linked immunosorbent assay. :: Biological and Pharmaceutical Bulletin. 2017 ; 巻 40, 番号 10. pp. 1767-1774.
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abstract = "Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle{\circledR} strain. Purified anti-GAA scFv, which yielded 2.56mg from 1L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078–1.25µg/mL) with precision (CV: ≤6.20{\%}) and reliability (recovery: 100.1–101.8{\%}) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.",
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AU - Nuntawong, Poomraphie

AU - Sakamoto, Seiichi

AU - Bhuket, Pahweenvaj Ratnatilaka Na

AU - Kohno, Toshitaka

AU - Kikkawa, Nao

AU - Rojsitthisak, Pornchai

AU - Shimizu, Kuniyoshi

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