In this study, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of estriol (E3) in milk was developed. Rabbit polyclonal antibody specific for E3 was raised by introducing a carboxyl spacer arm from bromoacetic acid of E3 as immunizing hapten. A heterologous ciELISA using a coating hapten containing a carboxyl spacer arm from 6-ketoestriol of E3. The assay exhibited the required sensitivity for detection of E3 with limit of detection of 0.07 ng mL −1 and a working range from 0.41 ng mL −1 to 15.78 ng mL −1. Good recoveries were obtained ranging from 83.2% to 125.8% for E3 spiked milk samples. A satisfactory correlation (R 2 = 0.994) between the results of ciELISA analysis and that of LC-MS/MS analysis indicated good accuracy and reproducibility of the proposed method, which was ideally suited as a rapid screening tool for routine E3 monitoring.

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