Deoxymiroestrol is the most potent phytoestrogen in the chromene group found in Pueraria candollei. The estrogenic activity of P. candollei, functions as hormone replacement therapy in postmenopausal women. This study established an enzyme-linked immunosorbent assay (ELISA) for quantifying deoxymiroestrol using fragment antigen-binding (Fab) antibody to improve analytical performance. Our ELISA specifically detected deoxymiroestrol in the range of 31.25–1,000 ng/mL. Intra- and inter-assay precision was 1.48%–7.11% and 0.58%–9.31%, respectively, the recovery of authentic deoxymiroestrol spiked into samples was 99.77%–101.61%, and the limit of detection was 30.80 ng/mL. The findings of P. candollei root bark determined using our developed ELISA with Fab antibody and an ELISA with anti-deoxymiroestrol polyclonal antibody were consistent (R2 = 0.9807). The developed ELISA can be applied to the quality control of deoxymiroestrol derived from P. candollei.
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