Digital Immunoassay for Rapid Detection of SARS-CoV-2 Infection 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 that can detect viral infection across various species. In this study, we developed a biosensor assay that adapted a monoclonal-antibody (mAb)-based blocking ELISA format into an Activate Capture + Digital Counting (AC + DC)-based immunoassay. The assay employs a photonic crystal (PC) biosensor, gold-nanoparticle (AuNP) tags, SARS-CoV-2 nucleocapsid (N) protein, and specific anti-N mAb to detect antibody responses in animals exposed with SARS-CoV-2. We demonstrated a simple 2-step 15-min test that was capable of detecting as low as 12.5 ng of antibody in controlled standard serum samples. Based on an evaluation of 176 cat serum samples with known antibody status, an optimal percentage of inhibition (PI) cut-off value of 0.588 resulted in a diagnostic sensitivity of 98.3% and a diagnostic specificity of 96.5%. The test is highly repeatable with low variation coefficients of 2.04%, 2.73%, and 4.87% across different runs, within a single run, and on a single chip, respectively. The test was further employed to detect antibody responses in multiple animal species as well as investigate dynamics of antibody response in experimentally infected cats. This test platform provides an important tool for rapid field surveillance of SARS-CoV-2 infection across multiple species.