Shengxin Lu

Preparation of Anti-Lomefloxacin Antibody and Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Lomefloxacin Residue in Milk

Lomefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a complicated instrumental method to detect lomefloxacin residue in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen of bovine serum albumin-lomefloxacin showed high sensitivity toward lomefloxacin with an IC50 value of 0.35 ppb in PBS buffer and was suitable to be used as a screening assay to detect lomefloxacin residue in food products. The ELISA (Enzyme-Linked ImmunoSorbant Assay) developed in this study was compared with a commercial ELISA kit and significant improvement was achieved in terms of sensitivity and specificity. The antibody prepared showed excellent specificity with certain cross-reactivity with only two drugs including norfloxacin (17.5%) and fleroxacin (8.8%) among commonly used (fluoro)quinolones. The assay measured drug residue in defatted milk spiked with lomefloxacin with an inter-assay coefficient of variation <22.2% and an intra-assay coefficient of variation <18.2%. The average recovery rates at 0.1, 0.3, 0.6, 1.0, and 2.0 ppb were in the range of 120–85% for inter-assay and in the range of 113–90% for intra-assay.

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.