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

Abstract

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.

Keywords:

• Antibody
• Chemiluminescence
• Danofloxacin
• Drug residue
• ELISA

Acknowledgments

This research was supported by the National Natural Science Foundation of China (No. 20675048), Shandong Natural Science Foundation (No. Y2008B31), and the National High-Tech Research and Developmental Program of China (863 Program, Nos. 07AA10Z435 and 2007AA06A407).

Notes

^a IC₅₀ value was the competitor concentrations where the absorbance value was decreased in half compared to that of group with no competitor. Data represent three separate experiments run on three different days.

^b Cross-Reactivity was determined by comparing the concentration of analyte required to produce a I/I ₀ = 50%. Results were expressed as a percentage relative to the figure for danofloxacin.

^a Intra-assay variation was determined by 5 replicates on a single day.

^b Inter-assay variation was determined by 4 replicates on 4 different days.