A colloidal gold immunochromatographic test strip based on mAbs anti-N protein to detect feline coronavirus

Feline coronavirus (FCoV) includes Feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV primarily affects the gastrointestinal system of cats, causing mild and self-limiting gastrointestinal symptoms. In contrast, FIPV infection leads to severe immune-mediated disease with rapid progression and a very high mortality rate. The occurrence of feline infectious peritonitis (FIP) is associated with mutations in the viral genome during FECV infection, which makes diagnosing FECV an effective measure for preventing FIP. This study aims to establish rapid and efficient detection methods to provide reliable diagnostic tools for disease prevention and treatment. In this study, the recombinant plasmid pCold I-FIPV-DF-2-N of the FIPV-DF-2 virus N protein was constructed. It was then expressed prokaryotically and purified. Using this plasmid, BALB/c mice were immunized, and two highly specific and stable monoclonal antibodies, mAbs 2G7 and mAb 3H5, were successfully prepared. mAb 2G7 recognizes the amino acid sequence 18RGRSNSRGRKN28 on the N protein, and mAb 3H5 recognizes the amino acid sequence 295GDQVKVTLTHTYYLPKDDAKTSQFLEQID323. We successfully developed a colloidal gold immunochromatographic test strip for FCoV detection using a double-antibody sandwich method. Experimental results showed that this test strip had a detection limit of 209.8 ng/mL for the recombinant pCold I-FIPV-DF-2-N protein; the overall agreement rate for detecting FCoV in 10 ascites samples, which tested positive by conventional PCR, was 70%; and there was no cross-reaction with feline parvovirus, feline calicivirus, and feline herpesvirus. The detection method can realize the rapid detection on site and provide a convenient detection method for pet clinics, animal shelters, etc.IMPORTANCEIn recent years, pets have become an indispensable part of people's lives. As companion animals, the number of pet cats in domestic families has increased year by year. According to the 2023 Pet White Paper, the number of pet cats in China has reached more than 65 million. Therefore, the prevention and treatment of infectious diseases in cats is becoming more and more important. Therefore, in this study, a double-antigen sandwich colloidal gold immunochromatographic assay was developed to detect feline coronavirus (FCoV). The detection of feline enteric coronavirus (FECV) excreted by cats can effectively implement treatment, reduce the coronavirus load, and further prevent the risk of FECV infection evolving into feline infectious peritonitis virus. This method is simple, rapid, and specific and can be used for the detection of FCoV. Therefore, this method is more suitable for pathogen diagnosis in the field than previous methods.