Directional evolution of progesterone receptor and development of an enhanced fluorescence polarization assay for screening of progestins in milk

Objective Fluorescence polarization immunoassay, a type of rapid and simple method, has been used to determine many veterinary drugs in food samples. However, no studies have reported the use of this method for the determination of progestins in food samples. In addition, the sensitivity of fluorescence polarization immunoassay is usually low, so a strategy capable of improving its sensitivity is desirable. Materials and Methods LEU718 in the binding cavity of the human progesterone receptor was replaced with glutamine (GLN) to produce a mutant based on virtual mutation and site-directed mutagenesis. A streptavidin/biotin based fluorescent tracer system was synthesized as a signal amplification system that was combined with the mutant to develop an enhanced fluorescence polarization assay for screening of progestins in milk. Results Molecule docking showed that the mutant exhibited improved recognition abilities for five progestins due to increased intermolecular forces. The method sensitivities for the five drugs were 22.6−68.5-fold greater (limits of detection of 0.14-0.6 ng/mL) in comparison with those of the conventional fluorescein-based signal system. As a result, the milk sample only needed a 10-fold dilution prior to analysis. In addition, the method sensitivities for the five drugs when using the mutant increased 2.2−5.2-fold in comparison with those of the parental receptor, confirming successful evolution. Conclusions This enhanced fluorescence polarization assay can be used as a practical tool for routine screening of progestins in milk. Under the guidance of the present study, similar single-site/multi-site evolution techniques for improving the recognition ability of a receptor or antibody may be reported in the future, and more signal amplification systems based on novel fluorophores may be created in the future.