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.

Development of Highly Sensitive Chemiluminescence Enzyme Immunostaining Assay to Determine Glycyrrhizin Content Using Anti-glycyrrhizin Monoclonal Antibody.

Licorice, the root of Glycyrrhiza spp., is used in a large number of herbal medicines, such as traditional Chinese medicines, Japanese Kampo medicines, and therapeutic drugs. Since glycyrrhizin (GL) is among the main components in licorice and exhibits numerous beneficial pharmacological activities, the content of GL directly affects biological activity. The quality control based on GL content is an important factor in ensuring biological activity; however, the content of GL in licorice varies depending on plant cultivation environment, genetic factors, and species type. Previously, we prepared an anti-GL monoclonal antibody (anti-GL mAb) and employed it in various immunochemical assays for quality control of licorice and licorice-based products. In this study, we employed the anti-GL mAb in chemiluminescence enzyme immunostaining (CLEIS) to develop a very simple, rapid, specific, and sensitive quality control assay for licorice products, with a limit of detection of 3.9 ng. Furthermore, the CLEIS assay enabled semiquantitative analysis of GL in Kampo medicines. Our results showed that multiple samples can be simultaneously analyzed using CLEIS, and it is a useful tool for determining GL content, as well as ensuring chemical quality control of licorice-containing products and herbal medicines.

Rapid magnetic particles-based enzyme immunoassay for the quality control of Glycyrrhiza spp. based on glycyrrhizin content.

Glycyrrhizin (GC) is a triterpenoid saponin isolated from the roots of Glycyrrhiza spp., a medicinal plant that is present in 70% of Kampo prescriptions. Since the GC content in Glycyrrhiza spp. affects its various pharmacological activities, Glycyrrhiza spp. is prescribed to contain at least 2% of GC in the Japanese pharmacopoeia, and its quality control based on GC content is required. In this study, a magnetic particles-based enzyme immunoassay (MPs-EIA) was developed using specific monoclonal antibody against GC (MAb 2H2) for the detection of GC in Glycyrrhiza spp. In this system, the immunoreaction time using primary and secondary antibodies was reduced by taking advantage of the wide surface area of magnetic particles (MPs) conjugated with GC by N,N'­carbonyldiimidazole (CDI)-mediated method. Optimization of MPs-EIA revealed that total assay time (~2 h) was reduced to over half of that of conventional indirect competitive enzyme-linked immunosorbent assay (ELISA) (~5 h). In addition, the GC concentration was detectable within the range from 97.7 to 781 ng/mL, with a limit of detection of 71.4 ng/mL. A series of further validation analyses support the reliability and accuracy of the developed MPs-EIA for the detection of GC in Glycyrrhiza spp. Since the present MPs-EIA overcomes the disadvantage of ELISA in terms of rapidity, it provides a useful approach for the effective quality control of Glycyrrhiza spp., especially when handling multiple samples.

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.

Simultaneous determination of glycyrrhizin and liquiritin in licorice roots and Kampo medicines by combination enzyme-linked immunosorbent assay using anti-glycyrrhizin and anti-liquiritin monoclonal antibodies.

Immunoassay systems using monoclonal antibodies (mAbs) are one of the most useful techniques in the analytical, biochemical, and clinical fields. In this study, a combination enzyme-linked immunosorbent assay (ELISA) using both anti-glycyrrhizin and anti-liquiritin mAbs (anti-GL/Liq mixture mAbs) was developed for quality control of licorice and its products. The combination ELISA demonstrated high sensitivity, reproducibility, and specificity for the total content of GL and Liq by a single assay. The developed ELISA was effective and useful as the first screening method in the selection of high-quality licorice from the Glycyrrhiza species and in confirming the quality of licorice-containing Kampo medicines.

Development of Double Eastern Blotting for Major Licorice Components, Glycyrrhizin and Liquiritin for Chemical Quality Control of Licorice Using anti-Glycyrrhizin and anti-Liquiritin Monoclonal Antibodies.

Licorice is utilized in various food industries around the world for seasoning agents, confectioneries, drinks, and functional foods. Glycyrrhizin (GL) and liquiritin (Liq) are major quality control chemical markers of licorice that have multifunctional bioactivities. Chemical quality control of licorice is important because its component profiles change depending environmental factors (climate, soil condition, and water deficit) and differences between species. Double eastern blotting using anti-GL and anti-Liq monoclonal antibodies was developed for more convenient, rapid, and specific quality control analysis of GL and Liq, respectively. Moreover, double eastern blotting was applied to investigate the immunohistochemical distributions of GL and Liq in the root of fresh licorice; the localization of both components was then clarified visually. This double eastern blotting technique for GL and Liq may serve as a powerful approach for visually determining the chemical quality of licorice.

Screening of Glycyrrhiza uralensis Fisch. ex DC. containing high concentrations of glycyrrhizin by Eastern blotting and enzyme-linked immunosorbent assay using anti-glycyrrhizin monoclonal antibody for selective breeding of licorice.

A rapid selection system was used to screen Glycyrrhiza uralensis plants containing high concentrations of glycyrrhizin (GC) by Eastern blotting using anti-GC monoclonal antibody (MAb). Chromatographic fingerprinting by Eastern blotting correlated with the GC concentration analyzed by enzyme-linked immunosorbent assay (ELISA). The roots of wild G. uralensis growing in Mongolia were analyzed by Eastern blotting to identify plants containing high concentrations of GC, and the GC concentration was confirmed by ELISA. G. uralensis plants cultivated in the greenhouse were also analyzed in the same manner. GC concentrations in wild G. uralensis roots and cultivated plants varied widely: between 0.06 and 9.36 percent dry weight (dw%). To confirm the homogeneity of GC concentrations in the cultivated plants, we monitored GC concentrations in the plants over 2 years. Although GC concentrations changed in two plants, they remained comparatively constant in the other five plants, suggesting that GC concentrations are genetically determined. To identify high GC-producing plants, 1025 plants were analyzed, and the highest concentration of GC was 5.36 dw%.