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.