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Sensitive immunoassays based on a monoclonal antibody for detection of marbofloxacin in milk
Meixia Luo1, Keyu Xing1, Zhen Guo2
1State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
Abstract:
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.
Insights
Highly sensitive monoclonal antibodies (mAbs) were developed for detecting Marbofloxacin (MBF) residues in milk. These antibodies enable effective screening using ELISA and immunochromatographic assays, ensuring food safety.
Area of Science:
- Veterinary Pharmacology
- Food Safety
- Immunochemistry
Background:
- Marbofloxacin (MBF) is a widely used veterinary fluoroquinolone antibiotic.
- Residues of MBF in animal-derived foods, like milk, pose a risk to consumer health.
- Sensitive detection methods are crucial for monitoring MBF contamination in food products.
Purpose of the Study:
- To produce and characterize monoclonal antibodies (mAbs) against Marbofloxacin (MBF).
- To develop and validate sensitive immunoassay methods for MBF detection in milk.
- To evaluate the efficacy of developed assays as screening tools for MBF residues.
Main Methods:
- Production of four monoclonal antibodies (mAbs) against Marbofloxacin (MBF).
- Indirect competitive (ic) ELISA was employed to determine antibody sensitivity (IC50, LOD) and cross-reactivity.
- Development of a visual colloidal gold-based immunochromatographic assay (ICA) for MBF detection.
Main Results:
- The M4E3 mAb demonstrated high sensitivity for MBF detection, with an IC50 of 0.07 ng/mL and LOD of 0.01 ng/mL.
- M4E3 showed significant cross-reactivity with other fluoroquinolones, indicating broad applicability.
- The developed icELISA method achieved recovery rates of 72.28%–129.19% in milk samples.
- A colloidal gold-based ICA with a cut-off value of 1 ng/mL was successfully developed for MBF detection in milk.
Conclusions:
- The developed M4E3 mAb is a highly sensitive reagent for Marbofloxacin (MBF) detection.
- Both indirect competitive ELISA and colloidal gold-based immunochromatographic assay are effective screening tools for MBF in milk.
- These methods contribute to ensuring the safety of animal-derived food products by detecting harmful antibiotic residues.
