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Single-Cell-Derived Rabbit mAb-Based Immunoassay for Simultaneous Detection of Florfenicol and Florfenicol Amine
Peipei Li1, Yantong Pan1, Yuan Li2
1State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, 100193Beijing, People's Republic of China.
Analytical Chemistry
|July 28, 2026
Summary
Researchers developed highly effective rabbit monoclonal antibodies (RmAbs) for detecting florfenicol (FF) and FF amine (FFA). These new antibodies offer superior affinity and stability, enabling rapid on-site detection in milk and eggs.
Area of Science:
- Immunotechnology
- Veterinary Pharmacology
- Analytical Chemistry
Background:
- Rabbits possess unique antibody structures and evolution mechanisms, suggesting potential for high-affinity antibody production against small molecules.
- Previous mouse monoclonal antibodies (MmAbs) against florfenicol (FF) and FF amine (FFA) showed limitations in affinity and stability.
Purpose of the Study:
- To discover and characterize novel rabbit monoclonal antibodies (RmAbs) with enhanced recognition profiles for FF and FFA.
- To develop a sensitive and rapid immunoassay for on-site detection of FF and FFA in food matrices.
Main Methods:
- Precise localization and sorting of single antibody-secreting cells (ASCs) from rabbits.
- Characterization of RmAbs using ELISA, molecular docking, and dynamic simulations.
- Development and validation of a lateral flow immunoassay (LFIA) for FF and FFA detection.
Main Results:
- Five RmAbs were discovered, with RmAb-A4# showing superior dual recognition of FF and FFA (IC50: 0.07 ng/mL and 0.16 ng/mL, respectively), significantly outperforming the best MmAb.
- RmAb-A4# exhibited enhanced halotolerance (18-5000 mM NaCl) and pH stability (pH 4-9) compared to MmAb-4F8.
- An LFIA based on RmAb-A4# achieved rapid, sensitive detection of FF and FFA in milk (0.22-0.17 μg/L) and eggs (0.74-0.78 μg/kg) without pretreatment or with minimal dilution.
Conclusions:
- Rabbit monoclonal antibodies can be generated with significantly higher affinity and stability for small molecules like FF and FFA.
- The developed RmAb-A4# is a promising candidate for sensitive, on-site detection of FF and FFA residues in food products.
- The single ASC sorting strategy provides a practical approach for discovering high-performance antibodies against small molecules.
