Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction‐derived hapten conjugate

Daidzin and its aglycone daidzein are major pharmacologically active compounds of soybean ( Glycine max ), kudzu ( Pueraria lobata ), and kwao kruea khao ( P . mirifica ). Pharmacological activities of daidzin are mediated by its more potent metabolites daidzein and equol; however, daidzin is the predominant compound found in these medicinal plants, and the efficacy and safety of equol depend on the amount of daidzin consumed. Objective To develop a specific monoclonal antibody (MAb)‐based indirect competitive enzyme‐linked immunosorbent assay (icELISA) for standardisation of daidzin content in herbal medicines or nutraceuticals. Methodology The Mannich reaction was used for the synthesis of a highly immunogenic conjugate between daidzin and a cationised carrier protein. Splenocytes of hyperimmunised mice were fused with myeloma cells to generate a hybridoma secreting antibody against daidzin. Results The icELISA showed high selectivity and acceptable sensitivity for daidzin determination (1.56–100 ng/mL) with high reproducibility (coefficients of variation were < 5%). The icELISA was a reliable analytical method for daidzin in Glycine max , Pueraria lobata and P . mirifica , for which daidzin recoveries from spiked samples were 98.99–104.94%. Daidzin content of these plant‐derived products determined using the icELISA were in close agreement with those determined by a HPLC‐UV method. Conclusion The icELISA is useful for specific daidzin determination because of its reliability, low cost, speed and high throughput. Copyright © 2015 John Wiley & Sons, Ltd.