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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, 100193 Beijing, People's Republic of China.
Analytical Chemistry
|June 3, 2026
Summary
Researchers developed novel rabbit monoclonal antibodies (RmAbs) for detecting florfenicol (FF) and FF amine (FFA). One antibody, RmAb-A4#, shows superior affinity and stability, enabling rapid on-site detection in milk and eggs.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Rabbit antibody production mechanisms may yield higher affinity antibodies for small molecules compared to mice.
- Developing sensitive and rapid detection methods for veterinary drug residues like florfenicol is crucial for food safety.
Purpose of the Study:
- To discover and characterize novel rabbit monoclonal antibodies (RmAbs) targeting florfenicol (FF) and its metabolite FF amine (FFA).
- To develop a sensitive and rapid immunoassay for on-site detection of FF and FFA in food matrices.
Main Methods:
- Single antibody-secreting cell (ASC) precise localization and sorting strategy was employed.
- Five RmAbs were discovered, with RmAb-A4# exhibiting high affinity and dual recognition of FF and FFA.
- Molecular docking and dynamic simulations were used to elucidate binding mechanisms.
- A lateral flow immunoassay (LFIA) was developed based on RmAb-A4#.
Main Results:
- RmAb-A4# demonstrated superior affinity (IC50 values of 0.07 ng/mL for FF, 0.16 ng/mL for FFA) compared to previously reported mouse monoclonal antibodies (MmAbs).
- RmAb-A4# exhibited enhanced halotolerance (18-5000 mM NaCl) and pH stability (pH 4-9).
- The developed LFIA achieved sensitive detection limits: 0.22 μg/L (FF) and 0.17 μg/L (FFA) in milk, and 0.74 μg/kg (FF) and 0.78 μg/kg (FFA) in eggs.
Conclusions:
- A practical single ASC sorting strategy successfully identified high-affinity RmAbs for small molecules.
- RmAb-A4# represents a significant advancement in antibody development for small molecule detection.
- The developed LFIA provides a sensitive and rapid tool for on-site monitoring of florfenicol residues in food products.
