Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

INTRODUCTION: Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. OBJECTIVE: To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. METHODOLOGY: This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. RESULTS: When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 µg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. CONCLUSION: The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Advanced quality assessment of Sanshishi (Gardenia jasminoides Ellis) and Kampo medicines using a monoclonal antibody against geniposide.

Gardenia jasminoides Ellis, a plant widely used in traditional medicine, is known for its array of biological activities. A key bioactive compound, geniposide (GE), an iridoid glycoside, significantly contributes to the medicinal properties of the plant, with potential side effects. Thus, a reliable and efficient method for GE detection is required to ensure the quality of medicinal-grade G. jasminoides Ellis. This study developed such a method by first synthesizing GE-bovine serum albumin conjugates to function as immunizing agents in mice. This led to the production of a monoclonal antibody (mAb 3A6) against GE from the fusion of splenocytes from immunized mice with myeloma cells (P3U1), resulting in a hybridoma that produces mAb 3A6. Thereafter, we developed a mAb 3A6-based indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA exhibited satisfactory sensitivity (0.391-12.5 µg/ml) and repeatability (coefficients of variation <10%). The accuracy of this method was validated through a spike-recovery assay (recovery of 101-112%). Furthermore, the icELISA was employed to determine the GE content in plant and Kampo medicine samples. The GE content positively correlated with those determined by high-performance liquid chromatography-ultraviolet. The proposed icELISA is rapid, cost-effective, and reliable for high-throughput GE detection in G. jasminoides Ellis, thereby contributing to the improved quality control and standardization of this valuable medicinal plant.

Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Quality assessment method for Chinpi, dried Citrus spp. peel and its derived Kampo medicines using specific monoclonal antibody against hesperidin.

INTRODUCTION: Hesperidin (hesperetin 7-rutinoside, HP), a flavonoid glycoside found in Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), has been reported to exert a variety of pharmacological effects. As the efficacies and qualities of their dried peel, Chinpi and its derived Kampo medicines can be evaluated by their HP contents, a method for HP detection must be developed. OBJECTIVES: To produce a specific monoclonal antibody against HP (mAb 5D12) to detect the HP contents in Japanese traditional medicines via indirect competitive enzyme-linked immunosorbent assay (icELISA). METHOD: BALB/c mice were immunised with many haptens of HP-bovine serum albumin (BSA) conjugates that were prepared using sodium periodate (NaIO4 ) to cause an immune response. In addition, conventional hybridoma techniques were utilised to generate mAb 5D12. RESULTS: The detection range of HP by the mAb 5D12-based icELISA was 1.56-25.0 ng/mL, with a detection limit of 1.12 ng/mL. The maximum coefficient of variation, as evaluated from the intra- and inter-assays, was <10.0%, and the percentages of recovery, as determined by the spike-recovery tests, were 105%-115%. Moreover, the HP content, which was obtained from the developed icELISA, correlated well with that obtained via high-performance liquid chromatography-ultraviolet (HPLC-UV). CONCLUSION: These validation analyses revealed that the established icELISA technique exhibited high precision and accuracy. Notably, this is the first report on the development of icELISA for the HP content-based quality control of Chinpi and its derived Kampo medicines.

Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06-625.00 ng/mL. The intra- and inter-assay precisions were 0.74-2.98% and 6.57-9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70-110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

Bioimprinting as a Receptor for Detection of Kwakhurin.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

Activated Carbon-Based Immunochromatographic Strip Test for the Rapid Qualitative Analysis of Swertiamarin and Sweroside.

BACKGROUND: Swertia japonica (S. japonica) is a medicinal plant that belongs to the Gentianaceae family. Several reports confirm the biological effects of the S. japonica extract. This plant is used mainly as a digestive stimulant, appetite stimulant, and gastrointestinal disease remedy in Japan. Secoiridoid glycosides are a group of compounds related to the beneficial effects of this plant. OBJECTIVE: We developed an immunochromatographic strip test for major secoiridoid glycosides, such as swertiamarin (SM) and sweroside (SS) detection. METHODS: We fabricated an immunoprobe using activated carbon as a reporter molecule and a monoclonal antibody against SM and SS (MAb D2) as a detection molecule. The test and control zones of the strip test contained SM-cBSA and Goat pAb anti-mouse IgM HRP conjugate, respectively. The immunoprobe reacted competitively with free SM and/or SS and immobilized SM-cBSA. The results were read and interpreted by the black spot intensity in the test zone. RESULTS: We succeeded in developing a strip test system with a detection limit (LOD) of 12.5 µg/mL. The selectivity and reliability evaluation revealed that the strip test is suitable for detecting SM and SS in S. japonica. The result was ready to be read in 30 min. CONCLUSIONS: This method can be a useful tool for the screening of biologically active S. japonica samples for further preparation of traditional medicine. HIGHLIGHTS: To the best of our knowledge, this is the first immunochromatographic strip test developed for the detection of SM and SS in S. japonica samples.

Lateral flow immunoassay for small-molecules detection in phytoproducts: a review.

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.

Competitive immunochromatographic test strips for the rapid semi-quantitative analysis of the biologically active bitter glycoside, amarogentin.

Amarogentin (AG), a biologically active secoiridoid glycoside, is responsible for the efficacy of Gentianaceae based medications. Thus, qualitative and quantitative analyses of AG are of significance for batch to batch quality control purposes. By conjugating colloidal gold nanoparticles with the AG-specific monoclonal antibody, MAb 1E9, we were able to develop a single-step competitive immunochromatographic assay (ICA) for simple quantification of the AG content in plant samples. With a limit of detection of 250 ng/mL, the analytical results were obtained after immersing the ICA test strip in the detection mixture for 15 min. This new ICA is superior to conventional ICAs as it is considerably faster due to the speed with which the test strips can be produced and the omission of the time-consuming preparation phase that was previously required to make the fiber pad. Moreover, our ICA only needs a small amount of analyte (20 µL).The reliability of the reported test strip was confirmed by comparing its semi-quantitative results with those obtained via an indirect competitive enzyme-linked immunosorbent assay (icELISA). The positive correlation between these methods (R2 = 0.984) indicated that this new ICA could be applied for the semi-quantitative analysis of the AG content in plant samples.

The colloidal gold nanoparticle-based lateral flow immunoassay for fast and simple detection of plant-derived doping agent, higenamine.

Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxyphenylacetaldehyde 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.

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham.

INTRODUCTION: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects. OBJECTIVES: This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species. METHODS: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chromatography with UV detector (HPLC-UV). The results of the two methods were compared for consistency checking. RESULTS: The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. CONCLUSION: The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to quantify swertiamarin and related compounds in Swertia japonica Makino.

INTRODUCTION: Swertia japonica Makino (S. japonica) has a long history of use as a folk medicine, and it is one of the three essential Japanese folk medicines. S.japonica has been reported to have various biological activities. The biologically active secoiridoid glycoside swertiamarin (SM) has been isolated from S. japonica. The efficacy of this plant is attributed to SM and related secoiridoid glycosides. To control the quality of S. japonica for medicinal use, a method for the determination of SM and other secoiridoid glycosides in the plant is needed. OBJECTIVE: To produce an anti-SM monoclonal antibody (MAb) and develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) for S. japonica standardisation and quality control. METHODOLOGY: SM was conjugated to cationised bovine serum albumin (cBSA), and the SM-cBSA conjugate was used to immunise BALB/c mice. Splenocytes from the immunised mice were then fused with SP2/0 myeloma cells to produce hybridoma cells that expressed anti-SM MAb. RESULTS: The developed icELISA was sufficiently sensitive and had a quantitative range of 0.78 to 12.5 µg/mL. Coefficients of variation below 10% indicated good repeatability. Recoveries in a spike and recovery assay ranged from 91.84% to 115.50%, which confirmed that the icELISA was accurate. The SM content measured using the icELISA was in agreement with the results of a high-performance liquid chromatography-ultraviolet (HPLC-UV) assay. CONCLUSION: The icELISA is suitable for the high-throughput analysis of SM and other secoiridoid glycosides in S. japonica. The method is fast, economical, and reliable for S. japonica quality control.

ELISA for the Detection of the Prohibited Doping Agent Higenamine.

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 ß2-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.

Kwakhurin-magnetic particles conjugates enable fast enzyme immunoassay for the detection of kwakhurin in Pueraria candollei.

INTRODUCTION: Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei-derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei-derived products has gained a sense of urgency in recent years. OBJECTIVE: This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products. MATERIAL AND METHOD: A rapid and sensitive immunoassay was developed with a combination of Kwa-specific monoclonal antibody (MAb 11F) and Kwa-magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time. RESULT: This novel MPs-based enzyme immunoassay (MPs-EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs-EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.

New 2-arylbenzofurans from the root bark of Artocarpus gomezianus and their α-glucosidase inhibitory activity.

Two new 2-arylbenzofurans, namely 13-O-methyllakoochin B (1) and artogomezianin (2), were isolated from the root bark of Artocarpus gomezianus, along with six known compounds (3-8). The structures of new compounds were determined by spectroscopic and chemical methods. All of the isolates were evaluated for their α-glucosidase inhibitory activity. Artogomezianin (2) and lakoochin A (3) exhibited strong α-glucosidase inhibitory activity with IC50 values of 18.25 and 26.19 µM, respectively, as compared with the positive control acarbose.

Different expression systems resulted in varied binding properties of anti-paclitaxel single-chain variable fragment antibody clone 1C2.

The binding properties of recombinant antibody fragments, such as affinity and specificity, determine their usefulness for therapeutic and analytical applications. Anti-paclitaxel single-chain variable fragment clone 1C2 (anti-PT scFv1C2) was expressed using Escherichia coli cell and Bombyx mori larvae expression systems. The binding characteristics of the scFvs were evaluated using indirect competitive ELISA. The linear range of binding between anti-PT scFv1C2 and paclitaxel was significantly different between the anti-PT scFv1C2s derived from E. coli (1.056-5.700 µg/ml) and B. mori (7.813-1000 ng/ml), which indicated that different expression systems resulted in different sensitivities for paclitaxel determination. In addition, the binding specificity of anti-PT scFv1C2 varied between expression systems. This finding implied that the expression system significantly affects the binding properties of recombinant antibodies, especially antibodies against low-molecular-weight targets.

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.

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.

Development of an indirect competitive immunochromatographic strip test for rapid detection and determination of anticancer drug, harringtonine.

Harringtonine (HT) is a natural compound, which is mainly produced by the genus Cephalotaxus, and has been clinically utilized in China for the treatment of acute leukemia and lymphoma. However, the amounts of HT in the Cephalotaxus species are very small; therefore, plant tissue cultures have been focused upon to enhance HT production. Qualitative/quantitative methods for HT detection are required to screen superior cell lines. We developed a one-step indirect competitive immunochromatographic assay (ICA) using colloidal gold nanoparticles conjugated with highly specific monoclonal antibodies against HT (MAb 1D2) for simple, rapid, and sensitive detection of HT in plant samples. This ICA can be completed in 15min after dipping the strip into analytes with a limit of detection of ∼313ng/mL. In developed ICA, fiber pad which is usually used for conventional ICA, was not used to shorten the time for preparing chromatographic strip, resulting in a decrease in the volume of valuable analytes (20µL). Considering simplicity, rapidity, and sensitivity of the developed ICA, this study could be applied to a fieldwork study for finding new natural resources containing HT.