Establishment of an indirect ELISA for feline coronavirus antibody detection and serotype discrimination

Abstract

Feline coronavirus (FCoV) is a highly contagious pathogen that is endemic to feline populations and is classified into two serotypes, I and II. Current diagnostic techniques are insufficient for distinguishing between these serotypes, which impedes effective surveillance and prevention efforts. In response to this limitation, we have developed an indirect enzyme-linked immunosorbent assay (ELISA) utilizing recombinant nucleocapsid (N) protein for the broad detection of FCoV, alongside receptor-binding domain (RBD) proteins specific to serotypes I (I-RBD) and II (II-RBD) for the purpose of serotype differentiation. The assay underwent systematic optimization, achieving high sensitivity (with detection limits of 1:64,000 dilution for N and I-RBD, and 1:32,000 for II-RBD) and specificity, exhibiting no cross-reactivity with feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). The reproducibility of the assay was validated, with intra- and inter-assay coefficients of variation remaining below 10 %. Clinical validation conducted on 123 feline serum samples indicated a seroprevalence of 73.17 %, with the serotype distribution comprising 91.11 % serotype I, 1.11 % serotype II, 2.22 % mixed infections, and 5.56 % cases that could not be typed. This ELISA represents a rapid, cost-effective, and field-deployable method for large-scale FCoV surveillance and serotyping, thereby contributing to enhanced feline health management and epidemiological research.