Digital Immunoassay for Rapid Detection of SARS-CoV-2 exposure in a Broad Spectrum of Animals

The ability of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to infect a wide-range of species raises significant concerns regarding both human-to-animal and animal-to-human transmission. There is an increasing demand for highly sensitive, rapid, and simple diagnostic assays capable of detecting viral infection across various species. In this study, we developed a biosensor assay based on a blocking ELISA (bELISA) immunoassay format. The assay employs a photonic crystal (PC) biosensor, gold-nanoparticle (AuNP) tags, SARS-CoV-2 nucleocapsid (N) protein, and specific anti-N monoclonal antibody (mAb) to detect antibody responses in animals exposed to SARS-CoV-2. Based on an evaluation of 162 cat serum samples with known antibody status, an optimal percentage of inhibition (PI) cut-off value of 0.5877 resulted in a diagnostic sensitivity of 97.80% and a diagnostic specificity of 98.67%. The assay demonstrated high repeatability with low variation coefficients across different conditions, ensuring consistent performance. Additionally, the assay successfully detected anti-N antibody responses in ferrets and deer as early as 14 days post-infection (DPI), and in cats infected with both Omicron (B.1.1.529) and B.1 D614G (B.1) variants as early as 7 DPI. These results highlight the assay's ability to detect infections early and reliably across species and its capability to identify multiple variants of SARS-CoV-2. This test platform provides an important tool for rapid field surveillance of SARS-CoV-2 infection across multiple species.