Serological analysis
microsphere immunoassay- MIA-« luminex »
Serum samples were tested using a microsphere immunoassay (MIA) for the presence of specific immunoglobulins against 5 coronavirus antigens: the SARS-CoV-2, SARS-CoV, NL63 and 229E nucleoproteins (NP) and the MERS-CoV spike (S) protein. Distinct MagPlex microsphere sets (Luminex Corp., USA) were bound to the recombinant viral proteins using the amine coupling kit (Bio-Rad Laboratories, USA) according to manufacturers’ instructions. The MIA procedure was performed as described previously with minor modifications (Vanhomwegen et al., 2017). Briefly, microsphere mixtures were sequentially incubated in the dark under constant shaking with a 1:400 dilution of serum samples, 4 μg/mL biotinylated protein A (Thermo Fisher Scientific, USA) and 2 µg/mL streptavidin-R-phycoerythrin (Life technologies, USA). After the final incubation, the median fluorescence intensity (MFI) of each antigen-coupled microsphere set was quantified for each sample using a BioPlex 200 instrument (Bio-Rad Laboratories). The seropositivity cut-off value was calculated for each antigen as three standard deviations above the mean MFI of ten negative control sera (sampled before 2019).
Serological analysis ( ELISA)
Cats sera were tested by an adapted version of the ID screen® SARS-CoV-2-N IgG indirect ELISA (IDvet, Grabels, France). The kit specifically detects anti-SARS-CoV-2 nucleocapsid antibodies. For this study, the conjugate supplied in the kit was changed to a multi-species conjugate to detect cats antibodies. Briefly, samples to be tested were diluted 1:20 in the sample dilution buffer and incubated for 45 min at room temperature. After washes, the multi-species conjugate, diluted in conjugate buffer, was added for 30 minutes at room temperature. The TMB substrate was added after the final washes, and the optical densities read at 450 nm after addition of stop solution. Calculation of ELISA cut-off value was established by the analysis of 16 negative cat control sera (sampled before 2019).   The mean (x) and standard deviation (SD) of these 16 negative. The cut-off value was taken as x + 3SD. OD values above this value were considered positive.