Identification of a novel N-terminal linear B-cell epitope on the FIPV nucleocapsid protein by a monoclonal antibody

Feline infectious peritonitis (FIP) is difficult to diagnose in clinical practice and has a high mortality rate, which underscores the need for reliable and standardized diagnostic tools. The nucleocapsid (N) protein, a major viral structural component, is a preferred target for serological and pathological assays because it is highly abundant in infected cells and has strong immunogenicity. In this study, a monoclonal antibody, designated mAb 4F3, was generated by immunizing mice with purified recombinant FIPV N protein fused with a His tag (His-N) and screening hybridomas via ELISA utilizing purified recombinant N protein fused with a glutathione S-transferase tag (GST-N). Characterization via ELISA, western blot, and indirect immunofluorescence assay (IFA) confirmed that mAb 4F3 binds effectively to the recombinant antigen and specifically recognizes the native N protein within FIPV-infected cells. Furthermore, the antibody exhibited no cross-reactivity with heterologous coronaviruses, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Fine epitope mapping identified the minimal linear epitope recognized by mAb 4F3 as 105FKDKIDGVFW114, located within the N-terminal domain (NTD) of the N protein. Sequence alignment further verified that this epitope is highly conserved (> 90%) among FCoV strains while sharing low homology with non-FCoV coronaviruses, demonstrating excellent viral specificity. We successfully generated and characterized mAb 4F3, an anti-FIPV N protein monoclonal antibody that is suitable for multiple detection platforms. We also precisely mapped its minimal linear epitope. These findings provide a valuable antibody resource and a molecular basis for the development of standardized FIP diagnostic techniques and for studies of the immunological functions of the N protein.