Higenamine is a natural benzyltetrahydroisoquinoline alkaloid produced by various plants. In the World Anti-Doping Agency report of 2020, higenamine is classified as a class S3 (selective and nonselective β2-agonist) prohibited substance. To minimize the problems resulting from the misuse of higenamine-containing products as well as from the abuse of doping agents in sport, numerous higenamine-detection methods have been investigated. In the present study, a monoclonal antibody against the (S)-enantiomer of higenamine was successfully produced and applied in the indirect competitive ELISA to detect the content of (S)-higenamine in plant samples and related products. By immunizing BALB/c mice with higenamine-BSA, the aforementioned monoclonal antibody was produced even when the hapten number, which was the higenamine molecules conjugated to the BSA molecule, was relatively low (approximately 4). The MAb was characterized and utilized in the established icELISA assay with a detectable range of 7.81 – 125 ng/mL. The assay limit of detection (LOD) was 4.41 ng/mL, indicating higher sensitivity than the conventional HPLC-UV methods. Various validation processes demonstrated that icELISA was precise, with the maximum CV (%) of the intra- and inter-assays of 11.58% and 10.18%, respectively. Moreover, the assay was accurate, with the recovery rates of spiked (S)-higenamine ranging from 82% to 113%, and sufficiently reliable for the detection of (S)-higenamine in various kinds of samples. Notably, the present study describes the first immunoassay for (S)-higenamine.

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Phytoproducts are involved in various fields of industry. Small-molecule (Mw < 900 Da) organic compounds can be used to indicate the quality of plant samples in the perspective of efficacy by measuring the necessary secondary metabolites and in the perspective of safety by measuring the adulterant level of toxic compounds. The development of reliable detection methods for these compounds in such a complicated matrix is challenging. The lateral flow immunoassay (LFA) is one of the immunoassays well-known for its simplicity, portability, and rapidity. In this review, the general principle, components, format, and application of the LFA for phytoproducts are discussed.

Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxypheny lacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM-γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM-γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 μL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples. K E Y W O R D S colloidal gold nanoparticles, higenamine, immunochromatographic test strips, lateral flow immunoassay, monoclonal antibody 1 | INTRODUCTION Higenamine (HM; 1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroiso quinoline-6,7-diol, Figure 1) is a benzyl tetrahydroisoquinoline alkaloid derived from various plant species, including Aconitum carmichaelii Debeaux. (Ranunculaceae), Nandina domestica Thunb., (Berberidaceae), Nelumbo nucifera Gaertn. (Nelumbonaceae), and Asarum heterotrophies F. Schmidt (Aristolochiaceae). 1,2 Given its chiral character, studies have revealed that the norcoclaurine synthase conformational selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to form the specific enantiomer, (S)-HM. 3-5 From a pharmacological perspective, HM is a partial

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...