Development of a new indirect ELISA test for the detection of 2 anti-feline coronavirus antibodies in cats

Feline coronavirus (FCoV) belongs to the family Coronaviridae and in-24 cludes two pathotypes, the less-virulent Feline Enteric Coronavirus (FECV), that replicates in the 25 enteric epithelial cells, and the Feline Infectious Peritonitis virus (FIPV), more-virulent, that repli-26 cates efficiently within monocytes/macrophages with systemic involvement and may cause Feline 27 Infectious Peritonitis (FIP), a progressive and often fatal disease. The diagnosis of FIP is complex 28 and requires different examinations. Among serological tests, the indirect Immunofluorescent An-29 tibody Test (IFAT), considered the gold standard, and the Enzyme-Linked Immunosorbent Assay 30 (ELISA) are the most widely used to detect FCoV antibodies. The aim of this work was the devel-31 opment of FCoVCHECK Ab ELISA, a new rapid indirect test for the detection of FCoV antibodies 32 in feline serum/plasma samples; Methods: FCoVCHECK Ab ELISA was developed after a meticu-33 lous set-up and cut-off analysis through several methods, including the Youden’s index and ROC 34 curve, to achieve the best test performance. It was validated by testing 110 feline sera (62 positives 35 and 48 negatives) against the reference IFAT and compared with two other rapid ELISA tests, 36 INgezim Corona Felino (Gold Standard Diagnostics) and ImmunoComb Feline Coronavirus (FCoV) 37 [FIP] Antibody Test Kit (Biogal); Conclusions: FCoVCHECK Ab ELISA agreed with IFAT at 96.4% 38 (93.5% sensitivity, 95% confidence interval (CI): 83.5% - 97.9%; 100% specificity, 95% CI: 90.8% - 39 100%), at 93.6% with ImmunoComb FCoV and 82.7% with INgezim Corona Felino. Intra- and inter-40 assay accuracy and precision gave coefficients of variation lower than 20%. Compared to IFAT, the 41 new assay correctly identifies positive and negative samples with a good correlation and, in addi-42 tion, it is simpler, faster and provides a less subjective reading of the results.