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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 25, 2026
Summary
Researchers developed highly effective rabbit monoclonal antibodies (RmAbs) for detecting florfenicol (FF) and FF amine (FFA). These novel antibodies offer superior affinity and stability, enabling rapid on-site immunoassay detection in food products.
Area of Science:
- Immunotechnology
- Analytical Chemistry
- Veterinary Pharmacology
Background:
- Rabbit antibody structure and evolution mechanisms may enable higher affinity antibodies for small molecules compared to mice.
- Previous mouse monoclonal antibodies (MmAbs) for 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 high affinity and distinct 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) using the lead RmAb.
Main Results:
- Five RmAbs were discovered, with RmAb-A4# showing superior dual recognition of FF and FFA (IC50s 0.07 ng/mL and 0.16 ng/mL).
- RmAb-A4# exhibited significantly higher affinity (2-463-fold) and enhanced halotolerance and pH stability compared to the best MmAb.
- A rapid LFIA was developed, achieving semiquantitative detection limits of 0.22 μg/L (FF) and 0.17 μg/L (FFA) in milk without pretreatment.
Conclusions:
- A practical single ASC sorting strategy successfully identified high-affinity RmAbs against small molecules.
- RmAb-A4# demonstrates exceptional performance, stability, and suitability for sensitive on-site immunoassay detection of FF and FFA.
- The developed LFIA provides a valuable tool for rapid, on-site food safety monitoring.
