2.3 SARS-CoV-2 immunoassays
All serum samples were first screened for qualitative detection of anti-SARS-CoV-2 antibodies using the Elecsys® Anti-SARS-CoV-2 test, an electrochemiluminescence immunoassay (Roche Diagnostics, Basel, Switzerland) using a Cobas e 411 instrument. The assay uses a recombinant protein representing the nucleocapsid (N) antigen in a double-antigen sandwich assay format, which favours the detection of high-affinity antibodies against SARS-CoV-2. All serum samples were then quantified using the Roche Elecsys® Anti-SARS-CoV-2 S test for the SARS-CoV-2 spike receptor binding domain. This test enables the determination of both the presence and level of antibodies against SARS-CoV-2 in serum. Both immunoassays were performed according to the manufacturer’s instructions. The efficiency of these kits was determined using standardised controls for each experiment. The results were reported as values of the cut-off index (COI, signal sample/cut-off). All samples were considered negative for anti-SARS-CoV-2 NP antibodies, with a COI value <1.0. As reported above, anti-SARS-CoV-2 NP antibodies in the sera were quantified using the Elecsys® Anti-SARS-CoV-2 S test. The total antibody content in the sample was expressed as U/mL, traceable to the Roche Diagnostics internal standard cut-off for anti-SARS-CoV-2 S, which was 0.8 U/mL.
Both tests were developed for human testing, but the double-antigen method is species independent, as was previously reported (Natale et al., 2021).
2.4 Microneutralisation test ( MTN) for SARS-CoV-2 and BCoV