Development and validation of a recombinant N protein-based indirect ELISA for serological detection of feline...

Feline infectious peritonitis (FIP), a fatal disorder driven by the feline infectious peritonitis virus (FIPV), has no clinically approved vaccine available to date-rendering prevention and early-stage diagnosis critical to its control. In this study, a feline infection model was developed via intraperitoneal inoculation of virus-laden ascitic fluid, with infection validation conducted using clinical observation, hematological testing, biochemical profiling, imaging examinations, histopathological analysis, and immunofluorescence assay. The viral nucleocapsid N gene underwent cloning and heterologous expression in Escherichia coli, followed by purification of the recombinant protein for subsequent antibody generation. With this purified recombinant protein serving as the immunogen, three monoclonal antibodies were successfully generated and comprehensively characterized; concurrently, three highly conserved linear B-cell epitopes were pinpointed and subjected to structural characterization. Subsequently, an indirect enzyme-linked immunosorbent assay targeting the nucleocapsid N protein was developed and optimized. This assay exhibited high sensitivity, specificity, and reproducibility, demonstrating 98 % concordance with immunofluorescence assay outcomes when tested on 50 clinical feline specimens. In summary, this study presents the development of a reliable FIP infection model, yields novel monoclonal antibodies, and delivers a robust serological assay for feline coronavirus detection. These findings provide valuable tools to inform future diagnostic protocols and control strategies for FIP.