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

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 2.56 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 enzymelinked 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.Key words Ganoderma lingzhi; ganoderic acid A; single-chain variable fragment antibody; Escherichia coli; enzyme-linked immunosorbent assay Antibodies are the most frequently used biological agents for routine diagnostics, therapeutics, and studies in various fields. Their applications to chemical analysis provide various advantages such as binding specificity and methodological simplicity that extend beyond conventional chromatographic techniques. The subjects of target molecules for immunoassays have been expanded from large molecules (i.e., protein, peptide, and DNA) to small molecules (herbicides, natural products, and phytohormones). As such, many monoclonal antibodies (mAbs) against small molecules (haptens), such as amikacin, 1) carbamazepine, 2) aflatoxins, 3) and daidzin, 4,5) have been produced to develop mAb-based immunoassays for qualitative and quantitative analyses, which were shown to be simple and convenient analytical methods. Because the cost of recombinant antibodies (rAbs) produced using Escherichia coli is much lower than that of antibodies produced using hybridoma cells or other animal cell lines, 6) immunoassays using E. coli-derived antibodies represent an economical approach for analytical applications. However, the E. coli-based production of rAbs against haptens is limited by an inability to retain rAb reactivity. Normally, refolding steps are required to recover the binding reactivity of the rAb, and the steps are determined by time-consuming trial-and-error procedures. The single-chain variable fragment (scFv) antibody is a simple and small arrangement of the functional rAb, in which the vari...

Establishment and Application of a Method for the Determination of Ganoderic Acid A

“…is study used synthetic immunogen to obtain antiganoderic acid A antibodies in the immune serum of experimental rabbits. An indirect competitive ELISA with good specificity and sensitivity for the detection of ganoderic acid A was constructed using synthetic coating antigen and antiganoderic acid A antibody, compared with the high-performance liquid chromatography adopted by Hongling Dong, Duanping Lu, and others [24,25], compared with the liquid chromatography-tandem mass spectrometry adopted by Liangqin Liu and Yongli Liu [26,27], and compared with the enzyme-linked immunosorbent assay method constructed by Sakamoto Seiichi, Gorawit Yusakul, and others [28][29][30]. In comparison, this method has a lower detection limit.…”

Construction, expression, and characterization of a single‐chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus

“…DNA template was resverse transcribed by total RNA that derived from hperimmune positive mice spleen after inoculation inactivated EMCV‐PV21 as antigen. Yusakul et al reported that they used SOE‐PCR producing a functional scFv antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderm lingzhi . Although the orientation of VH‐link‐VL or the VL‐linker‐VH can express functional scFv, the prokaryote expression of VL‐linker‐VH scFv is 20 times higher than in the opposite direction scFv .…”

Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

“…Additionally, Rosetta (or Transetta) hosts supplemented with tRNAs of four E. coli rare codons are also candidates that can be used to improve the low expression levels of scFv to some extent (Sharma et al., 2014; Wang, Dong, et al., 2016). Certain engineered strains, such as CyDisCo (Gaciarz & Ruddock, 2017) and Shuffle (Ismail et al., 2015; Yusakul et al., 2017) also play a role in maintaining the formation of disulfide bonds in scFv (Kang & Seong, 2020).…”