Development of an indirect competitive chemiluminescent enzyme immunoassay for ethanamizuril residue detection in eggs and feed

Ethanamizuril (EZL), a newly developed antiparasitic drug, is expected to be widely used in poultry production in the future, making monitoring its residues in food imperative. For a more sensitive method for detecting EZL residues in eggs and feed, this study developed an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for EZL detection. This assay consisted of EZL-ovalbumin and an HRP-labelled anti-EZL monoclonal antibody as the capture antigen and detection antibody, respectively, and luminol as the chemiluminescent substrate. By optimizing the reaction conditions, the ic-CLEIA achieved a sensitivity (half-maximal inhibitory concentration, IC50) of 0.12 ng/mL and a linear range (IC20–IC80) of 0.02–0.92 ng/mL. Both the intra-assay and inter-assay coefficients of variation were less than 12%, demonstrating excellent reproducibility and high agreement with HPLC–MS/MS results. The ic-CLEIA demonstrates high sensitivity, good accuracy, strong specificity, and rapidity, providing an effective technical approach for large-scale EZL sample monitoring.