The virion-containing fraction was then collected and washed by sterile PBS twice with ultracentrifugation at 28,000 for 2 h at 4 C to remove any residual sucrose. of cell lines originating from different animals , and it can also infect a number of animal species including ferrets, hamsters, macaques, mice, monkeys, rabbits, and tree shrews, in laboratory settings [3,4], it is important to understand the Amyloid b-Protein (1-15) possibility of it causing natural infections in animals. Cases of SARS-CoV-2 infections in captive animals and pets, including dogs, domestic cats, and large felines such as tigers, lions, snow leopards, pumas and cougars, ferrets, minks, as well as gorillas were reported in the following places: Hong Kong, Belgium, China, the USA, the Netherlands, France, Spain, Germany, Russia, Denmark, the UK, Amyloid b-Protein (1-15) Japan, South Africa, Italy, Sweden, Chile, Canada, Brazil, Greece, Argentina, Lithuania, Switzerland, Mexico, Slovenia, Estonia, Bosnia and Herzegovina, Amyloid b-Protein (1-15) Poland, and Latvia . In Valencia, Spain, around 1.5% of companion ferrets were found to be seropositive to SARS-CoV-2 in 2020 . While a serological survey in Lombardy, Italy showed that only around 1% of free-ranging stray colony and abandoned shelter domestic cats in the region were seropositive to SARS-CoV-2 during the pandemic , another study in Wuhan, China demonstrated that the seroprevalence of SARS-CoV-2 in domestic cats from animal shelters, pet hospitals, or COVID-19 patients families could be as high as 14.7% . Moreover, the possibility of SARS-CoV-2 transmission among domestic cats was also demonstrated in laboratory settings [9,10]. In addition, large scale outbreaks were reported in mink farms in Europe and North America . In particular, at least 15,000 farmed minks died of COVID-19 in Michigan, Utah and Wisconsin in the USA ; and a wild mink in Utah was found to be positive for the disease . Around 17 million farmed minks were culled in Denmark to prevent viral spreading . In addition, worryingly, genomic studies in the Netherlands showed evidence of two-way COVID-19 transmission between humans and minks . Therefore, there is a need to identify COVID-19 infections in animals, which would be undeniably important in the subsequent establishment of preventive measures. The most widely used method for laboratory diagnosis of COVID-19 is quantitative reverse transcriptionCpolymerase chain reaction (qRTCPCR). However, qRTCPCR can only detect the infection in its acute stage. As infections in animals can easily go unnoticed because they do not complain of fever, sore throat, etc., infections in the acute stage may be missed. Therefore, antibody detection is an excellent way of confirming the disease even after the animal has recovered from the illness. Since SARS-CoV-2 can infect a variety of animals, the optimal serological test would be one which is able to detect the SARS-CoV-2 antibody from different kinds of animal species. Currently a few in-house-developed or commercial multi-species indirect enzyme-linked immunosorbent assays (ELISAs) are available [15,16], where detection of the host antibodies relies on the use of HNPCC a multi-species secondary antibody which recognises mammalian immunoglobulin G (IgG); however, utility of this multi-species secondary antibody has not yet been validated for Amyloid b-Protein (1-15) all mammalian species . In this study, we used the recombinant nucleocapsid (N) protein of SARS-CoV-2 and a monoclonal antibody (mAb) against it to develop a competitive ELISA (cELISA) for SARS-CoV-2 infection in virtually all kinds of animals. The assay developed was then validated for its analytical performance using positive serum samples from a laboratory guinea pig (for 20 min at 4 C to remove cellular debris. The clarified supernatant was then inactivated with formaldehyde at a final concentration of 0.4% (overnight at 4 C. Subsequently, the supernatant was discarded, and the virion pellet was washed twice with sterile phosphate-buffered saline (PBS; Gibco). The concentrated inactive virus was resuspended using 10% polyethylene glycol (PEG)-8000 (for 30 min and the PEG-precipitated virions were resuspended with ice-cold sterile PBS. Resuspended virions were then added to a 40C60% sucrose gradient (in 0.22 m filter-sterilized PBS) drop by drop, followed by ultracentrifugation at 28,000 for 2 h at 4 C. The virion-containing fraction was then collected and washed by sterile PBS twice with ultracentrifugation at 28,000 for 2 h at 4 C to remove any residual sucrose. Lastly, the virion pellet was resuspended in PBS and viral concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). 2.2.3. Animal Immunization Ten guinea.