Sensitive immunoassays based on a monoclonal antibody for detection of marbofloxacin in milk

• Published in: Journal of dairy science Vol. 103; no. 9; pp. 7791 - 7800
• Main Authors: Luo, Meixia, Xing, Keyu, Guo, Zhen, Guo, Debin, Lai, Weihua, Peng, Juan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2020
• Subjects: Animals; Anti-Bacterial Agents - analysis; Antibodies, Monoclonal - immunology; Cattle; colloidal gold-based immunochromatographic assay; Cross Reactions; Drug Residues - analysis; Enzyme-Linked Immunosorbent Assay - veterinary; Female; Fluoroquinolones - analysis; Gold Colloid - chemistry; Immunoassay - methods; Immunoassay - veterinary; indirect competitive ELISA; marbofloxacin; Mice; Mice, Inbred BALB C; Milk - chemistry; monoclonal antibody; colloidal gold-based immunochromatographic assay; marbofloxacin; monoclonal antibody; indirect competitive ELISA
• ISSN: 0022-0302; 1525-3198
• DOI: 10.3168/jds.2019-18108

Marbofloxacin (MBF) is a key class of synthetic fluoroquinolone antibiotic that is commonly used as a veterinary drug. However, excess residue of MBF in animal-derived food samples, such as milk, is harmful to consumers. In this study, 4 mAb against MBF, namely, M4E3, M7A6, M3C7, and M5C6, were produced. Indirect competitive (ic) ELISA revealed that the M4E3 exhibited the highest sensitivity with a half-maximal inhibitory concentration (IC50) of 0.07 ng/mL and a limit of detection of 0.01 ng/mL for detection of MBF. The results of the cross-reactivity experiment revealed that the M4E3 could detect lomefloxacin, ofloxacin, fleroxacin, pefloxacin, danofloxacin, and enrofloxacin sensitively with IC50 of 0.02, 0.07, 0.18, 0.27, 0.30, and 0.32 ng/mL, respectively. Meanwhile, a cross-reactivity experiment showed that the M4E3 had a crossover rate of more than 20% with these fluoroquinolone analogs. A weak crossover rate below 1.11% was observed with 14 other fluoroquinolone analogs. The recovery rate of MBF in milk ranged from 72.28% to 129.19%, which showed that the developed indirect competitive ELISA was effective in detecting MBF in milk. Furthermore, a visual colloidal gold-based immunochromatographic assay was developed for detecting MBF with a cut-off value of 1 ng/mL in both phosphate-buffered saline and a milk sample by using this mAb. Given this sensitive mAb, both indirect competitive ELISA and colloidal gold-based immunochromatographic assay could be effective screening tools for detection of MBF in milk.