No 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...

Tubulin polymerization is an important target for anticancer therapies. Even though the potential of Ganoderma triterpenoids against various cancer targets had been well documented, studies on their tubulin polymerization-stimulating activity are scarce. This study was conducted to evaluate the effect of Ganoderma triterpenoids on tubulin polymerization. A total of twenty-four compounds were investigated using an in vitro tubulin polymerization assay. Results showed that most of the studied triterpenoids exhibited microtuble-stabilizing activity to different degrees. Among the investigated compounds, ganoderic acid T-Q, ganoderiol F, ganoderic acid S, ganodermanontriol and ganoderic acid TR were found to have the highest activities. A structure-activity relationship (SAR) analysis was performed. Extensive investigation of the SAR suggests the favorable structural features for the tubulin polymerization-stimulating activity of lanostane triterpenes. These findings would be helpful for further studies on the potential mechanisms of the anticancer activity of Ganoderma triterpenoids and give some indications on the design of tubulin-targeting anticancer agents.

Ganoderma is a genus of medicinal mushroom traditionally used for treating various diseases. Ganoderic acid A is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma species. Herein, we produced a highly specific monoclonal antibody against ganoderic acid A (MAb 12?A) and developed an indirect competitive ELISA for the highly sensitive detection of ganoderic acid A in Ganoderma lingzhi, with a limit of detection of 6.10?ng/mL. Several validation analyses support the accuracy and reliability of the developed indirect competitive ELISA for use in the quality control of Ganoderma based on ganoderic acid A content. Furthermore, quantitative analysis of ganoderic acid A in G. lingzhi revealed that the pileus exhibits the highest ganoderic acid A content compared with the stipe and spore of the fruiting body; the best extraction efficiency was found when 50?% ethanol was used, which suggests the use of a strong liquor to completely harness the potential of Ganoderma triterpenoids in daily life.