Development of a Multispecies Double-Antigen Sandwich ELISA Using N and RBD Proteins to Detect Antibodies against SARS-CoV-2

SIMPLE SUMMARY: SARS-CoV-2 infects humans and several animals, but infection of companion and zoo animals occurs mainly from humans through the aerosol route. Constant monitoring of the infection in animals is essential because of their close contact with humans, which increases the risk of infection and the potential for a surge of new viral variants. Therefore, it is of relevance to develop economical, fast, and effective diagnostic tests that are able to detect previous infections in multiple animal species. This study aimed to produce a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) using N and RBD proteins. The results show that this assay is able to detect antibodies against SARS-CoV-2 in serum samples from cats, dogs, guinea pigs, rats, tigers, and humans. ABSTRACT: SARS-CoV-2 infects humans and a broad spectrum of animal species, such as pets, zoo animals, and nondomestic animals. Monitoring infection in animals is important in terms of the risk of interspecies transmission and the emergence of new viral variants. Economical, fast, efficient, and sensitive diagnostic tests are needed to analyze animal infection. Double-antigen sandwich ELISA has the advantage of being multispecies and can be used for detecting infections caused by pathogens that infect several animal hosts. This study aimed to develop a double-antigen sandwich ELISA using two SARS-CoV-2 proteins, N and RBD. We compared its performance, when using these proteins separately, with an indirect ELISA and with a surrogate virus neutralization test. Positive and negative controls from a cat population (n = 31) were evaluated to compare all of the tests. After confirming that double-antigen sandwich ELISA with both RBD and N proteins had the best performance (AUC= 88%), the cutoff was adjusted using positive and negative samples from cats, humans (n = 32) and guinea pigs (n = 3). The use of samples from tigers (n = 2) and rats (n = 51) showed good agreement with the results previously obtained using the microneutralization test. Additionally, a cohort of samples from dogs with unknown infection status was evaluated. These results show that using two SARS-CoV-2 proteins in the double-antigen sandwich ELISA increases its performance and turns it into a valuable assay with which to monitor previous infection caused by SARS-CoV-2 in different animal species.