Development of an Indirect Chemiluminescent Competitive ELISA to Detect Danofloxacin Residues in Milk

In this study, a sensitive and specific indirect chemiluminescent enzyme-linked immunosorbance assay method (CL-ELISA) was developed to detect danofloxacin residue in milk. The influence of experimental factors on the results of the method was investigated, including the concentration of coating antigen and primary antibody, the dilution factor of the IgG-HRP antibody, as well as the type of microtiter plate. Under the optimized condition, the linear working range and IC₅₀ value were determined to be 0.025–0.5 ng · mL^?1 and 0.1 ng · mL^?1, respectively. Compared with traditional colorimetric ELISA, the CL-ELISA developed in this research demonstrated significant improvement in terms of sensitivity (20 times improvement) and linear range (25 times wider). The developed method was applied in detection of danofloxacin residue in milk and satisfied recovery rates of 92.6–105.2% and coefficient variations of 2.4–9.5% were obtained, demonstrating that the proposed method was accurate and reliable. Moreover, there was almost no any meaningful cross-reactivity with nine other (fluoro)quinolones, indicating its high specificity.