High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei

• We developed ELISA with very high sensitivity for miroestrol determination.
• Developed ELISA could be applied without other chemical interference as HPLC-UV.
• Developed ELISA could be applied for high throughput purpose.
• Developed ELISA could determine in samples without complex pre-treatment.

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73–3000 ng mL−1, which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80–104.37% and 98.31–106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R2 = 0.9996) (Yusakul et al. [18]) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695 ± 0.037–12.108 ± 0.285 μg g−1 dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.

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