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

• Published in: Biological & pharmaceutical bulletin 2017/10/01, Vol.40(10), pp.1767-1774
• Main Authors: Yusakul, Gorawit, Nuntawong, Poomraphie, Sakamoto, Seiichi, Bhuket, Pahweenvaj Ratnatilaka Na, Kohno, Toshitaka, Kikkawa, Nao, Rojsitthisak, Pornchai, Shimizu, Kuniyoshi, Tanaka, Hiroyuki, Morimoto, Satoshi
• Format: Article
• Language: English
• Subjects: Bacteria; Cost-Benefit Analysis; Cytoplasm; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - economics; Enzyme-Linked Immunosorbent Assay - methods; Enzymes; Escherichia coli; Escherichia coli - genetics; Ganoderic acid; ganoderic acid A; Ganoderma lingzhi; Heptanoic Acids - immunology; Immunoassay; Immunoassays; Lanosterol - analogs & derivatives; Lanosterol - immunology; Linearity; Purification; Quantitative analysis; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; single-chain variable fragment antibody
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b17-00531

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.