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

Introduction: 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.