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

Abstract

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.

Keywords:

• Antibody
• Cross-reactivity
• Drug residue
• Immunoassay
• Lomefloxacin

Abbreviations:

• BSA: bovine serum albumin
• cBSA: cationized BSA
• cOVA: cationized OVA
• cFA: complete Freund's adjuvant
• Da: unit of molecular mass Dalton
• DMF: N,N-dimethyl formamide
• EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
• IC₅₀: concentration at 50% inhibition
• iFA: incomplete Freund's adjuvant
• MRL: maximum residue level
• NHS: N-hydroxysuccinimide
• OPD: o-phenylendiamine
• OVA: ovalbumin
• PBS: phosphate buffered saline
• PBST: phosphate buffered saline containing 0.05% Tween 20
• SAS: saturated ammonium sulfate

Acknowledgments

This research was supported by National Natural Science Foundation of China (No. 20675048), National High-Tech Research and Development Program of China (863 Program, No. 07AA10Z435, No. 2007AA06A407), and National Basic Research Program of China (973 Program, No. 2007CB936602).

Notes

^a MA method: mixed anhydride method.

^b EDC antibody: the antibody prepared by EDC method.

^c MA antibody: the antibody prepared by mixed anhydride method.

^d EDC/MA antibody: the antibody prepared by using EDC immunogen and MA immunogen alternatively in immunization procedure.

^a IC₅₀ values in ppb.

^b Cross-reactivity was determined by comparing the concentration of analyte required to produce a B/B₀  = 50%. Results were expressed as a percentage relative to the figure for lomefloxacin.

^a Inter-assay variation was determined by 6 replicates on 4 different days.

^b Intra-assay variation was determined by 14 replicates on a single day.