<scp>HRP</scp> ‐Labeled Monoclonal Antibody Method for Detecting Residual Flurogestone Acetate in Food Products

ABSTRACT Flurogestone acetate (FGA) is a highly effective synthetic progesterone analog extensively utilized to regulate estrus synchronization, enhance superovulation, and improve the economic productivity of female livestock. However, FGA is associated with potential toxicity, and its improper application can lead to environmental residues, resulting in adverse effects on both humans and animals. These residues pose a significant threat to human health, underscoring the critical need for the development of rapid and accurate detection methods. In this study, the complete antigen of FGA was successfully synthesized, and a high‐affinity monoclonal antibody against FGA was developed. Utilizing this antibody, a direct competitive ELISA (dc‐ELISA) method based on HRP and monoclonal antibody coupling was established for the efficient detection of FGA residues in milk. The assay exhibits a validated quantitation range of 0.0034–0.97 ng mL −1 (IC 50 = 0.058 ng mL −1 ; LOD = 0.0013 ng mL −1 ). In milk (20‐fold dilution to mitigate matrix effects), recoveries at 0.2, 0.5, and 0.8 ng mL −1 were 95.63%–116.44%. The anti‐FGA monoclonal antibody displayed no cross‐reactivity ≥ 10% ( n = 6) among tested analogs (highest: medroxyprogesterone acetate, 7.46%). Samples whose calculated concentrations exceed the upper limit of 0.97 ng mL −1 are reported as positive (above range) without assigning a numeric value. These attributes support the use of this HRP‐labeled dc‐ELISA as a rapid screening method for FGA residues in milk. In conclusion, this study presents a simple and effective method for detecting residual flurogestone acetate in animal‐derived foods. Compared to indirect competitive ELISA (ic‐ELISA), the developed method significantly reduces the reaction time and enhances the detection efficiency.