Development of a natural porcine-estrogen-receptor-based pseudo immunoassay for screening of 13 estrogens in milk and studying its recognition mechanism

Background There have been some immunoassays reported for screening of the residues of estrogens in milk, but these methods can only determine one drug because of the limited recognition abilities of the used antibodies. Due to the broad specific recognition ability, receptor can be used as “special antibody” to develop pseudo immunoassay. However, there has been no article reporting the use of estrogen receptor for determination of estrogens in foods of animal origin so far. Furthermore, the recognition mechanism of estrogen receptor for estrogens have not been thoroughly studied. Results In this study, a type of magnetic probe based on 17β-estradiol was synthesized that was used to produce the natural porcine estrogen receptor with ovarian as the source tissue. Then its recognition mechanisms for 13 estrogens were studied based on the optimal homological model. Results showed it only interacted with estrogens but did not interact with other steroid hormones. Then it was utilized as a broad specific recognition reagent to develop a direct competitive method on 96-well microplate for detection of the 13 drugs in milk. Due to the utilization of streptavidin-biotin labeled horseradish peroxidase as signal amplification system, the sensitivities for the 13 estrogens (limits of detection 0.007-0.0573 ng/mL) were improved for 33-100 folds in comparison with the use of conventional horseradish peroxidase system (limits of detection 0.31-3.53 ng/mL). This method showed no cross-reactivity with other steroid hormones, consistent with the docking result. Significance This is the first study reporting the use of magnetic probe to produce natural receptor from animal tissues, and this is also the first study reporting a natural estrogen-receptor-based method for multi-determination of estrogens in food sample. With the guidance of this study, more receptors and related analytical methods for other drugs or small molecules maybe are reported in the future.