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.

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.

An indirect competitive enzyme-linked immunosorbent assay toward the standardization of Pueraria candollei based on its unique isoflavonoid, kwakhurin.

Kwakhurin (Kwa) is a plant secondary metabolite solely present in Pueraria candollei var. mirifica (P. candollei), which has long been used as a Thai traditional herb for estrogen replacement therapy. Recently, health hazards have arisen in Japan regarding P. candollei-derived products containing potent estrogenic compounds. Therefore, the development of standardization methods for P. candollei materials is an urgent problem requiring resolution. The enzyme-linked immunosorbent assay (ELISA) is an effective analytical technique because it enables the development of sensitive and specific assays of the target compound through antigen-antibody reaction. Here, we produced a monoclonal antibody against Kwa (MAb 11F) by immunizing Kwa-bovine serum albumin (BSA) conjugates prepared using an N,N'-carbonyldiimidazole (CDI) mediated method. Stability and cross-reactivity tests of MAb 11F revealed that the MAb 11F is stable for at least 4 months at 4 °C and is highly specific to Kwa. The detectable concentration range of an indirect competitive ELISA (icELISA) using MAb 11F exhibited values of 1.53-48.8 ng/mL with the limit of detection (LOD) of 1.13 ng/mL. Validation analyses revealed that the developed icELISA is precise, accurate, and reliable enough to be applied to P. candollei-derived samples and products for their standardization.