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

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University School of Pharmacy, Walailak University

• Nuntawong Poomraphie

  Faculty of Pharmaceutical Sciences, Chulalongkorn University

• Sakamoto Seiichi

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

• Ratnatilaka Na Bhuket Pahweenvaj

  Faculty of Pharmaceutical Sciences, Chulalongkorn University

• Kohno Toshitaka

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

• Kikkawa Nao

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

• Rojsitthisak Pornchai

  Faculty of Pharmaceutical Sciences, Chulalongkorn University

• Shimizu Kuniyoshi

  Department of Agro-Environmental Sciences, Graduate School of Agriculture, Kyushu University

• Tanaka Hiroyuki

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

• Morimoto Satoshi

  Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University

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 2.56 mg from 1 L 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: 0.078–1.25 µg/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.

収録刊行物

• Ｂｉｏｌｏｇｉｃａｌ　＆　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｂｕｌｌｅｔｉｎ

  Ｂｉｏｌｏｇｉｃａｌ　＆　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｂｕｌｌｅｔｉｎ 40 (10), 1767-1774, 2017

  公益社団法人 日本薬学会

キーワード

• <i>Ganoderma lingzhi</i>
• ganoderic acid A
• single-chain variable fragment antibody
• <i>Escherichia coli</i>
• enzyme-linked immunosorbent assay

詳細情報

• CRID: 1390001204633078400
• NII論文ID: 130006110575
• NII書誌ID: AA10885497
• DOI: 10.1248/bpb.b17-00531
• ISSN: 13475215
• ISSN: 09186158
• NDL書誌ID: 028541368
• PubMed: 28966249
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I028541368
• Web Site: https://www.jstage.jst.go.jp/article/bpb/40/10/40_b17-00531/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2018212730
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可