Two-step single B cell technology for high-affinity rabbit monoclonal antibodies: development of a highly sensitive immunoassay for monensin in raw milk

  • Food Chem. 2026 Jun 15:514:149116. doi: 10.1016/j.foodchem.2026.149116.
Jingjie Huang  1 ,  Ao Lin  2 ,  XinXin Ma  3 ,  Yanzhe Qing  2 ,  Yani Gu  2 ,  Xiaole Pan  3 ,  Jiancheng Li  4
Affiliations
  • 1. College of Veterinary Medicine, National Key Laboratory of Veterinary Public Health and Safety, Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing 100193, PR China; Hainan Technology Innovation Center for Food Safety Surveillance and Detection, Sanya Institute of China Agricultural University, Hainan 572000, PR China.
  • 2. College of Veterinary Medicine, National Key Laboratory of Veterinary Public Health and Safety, Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing 100193, PR China.
  • 3. Hainan Technology Innovation Center for Food Safety Surveillance and Detection, Sanya Institute of China Agricultural University, Hainan 572000, PR China.
  • 4. College of Veterinary Medicine, National Key Laboratory of Veterinary Public Health and Safety, Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing 100193, PR China; Hainan Technology Innovation Center for Food Safety Surveillance and Detection, Sanya Institute of China Agricultural University, Hainan 572000, PR China. Electronic address: [email protected].
PMID: 41956048 DOI: 10.1016/j.foodchem.2026.149116
Abstract

The feed additive monensin (MON) poses consumer health risks, requiring sensitive detection. Thus, we developed a two-step endoplasmic reticulum-directed antibody-secreting cell selection (ER-DAS) platform for rapid isolation of high-affinity monoclonal antibodies, overcoming the key limitations of the lengthy screening cycles of hybridoma technology and low yield of conventional single B-cell approaches. The ER-DAS achieves 48-h isolation of MON-specific antibody-secreting cells (ASCs) through integrated fluorescence-activated cell sorting (FACS) enrichment and nanopore-array imaging technology. Using this platform, a novel high-affinity recombinant rabbit monoclonal antibody (RmAb) was successfully produced. Molecular docking showed that RmAb 3G interacts with MON primarily through π-π stacking and hydrogen bonding. A convenient and cost-effective Immunoassay based on RmAb 3G exhibited an IC50 of 0.55 ng mL-1, lower than previously reported antibodies, exceptional salt and organic solvent tolerance, and 88.7-97.1% spike recoveries in raw milk. This work provides a streamlined workflow for efficient antibody development in food safety monitoring.

Keywords
Immunoassay; Monensin; Rabbit monoclonal antibody; Raw milk; Recognition mechanism; Single B cell technology.
Products