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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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® 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.

Original languageEnglish
Pages (from-to)1767-1774
Number of pages8
JournalBiological and Pharmaceutical Bulletin
Volume40
Issue number10
DOIs
Publication statusPublished - Jan 1 2017

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

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