Development of novel Competitive Enzyme-linked immunosorbent assays to detect SARS-CoV-2-specific antibodies in animals
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19-causing virus, is a zoonotic pathogen. There is concern about the virus spilling over from humans into wildlife species, which may then serve as reservoirs for future infection of humans and other animals. Furthermore, the level of exposure of potentially susceptible wildlife species is currently not known. There is, therefore, an urgent need to develop a single test that could be used for the serosurveillance of multiple wildlife species for exposure to SARS-CoV-2. Although there are serological techniques to detect the exposure of humans to the virus, few assays have the capacity to detect antibodies in a wide variety of species. Here, I describe the development of a competitive enzyme-linked immunosorbent assay (cELISA) to detect SARS-CoV-2 antibodies in mammals for which species-specific reagents are not available. Therefore, cELISAs were developed to detect SARS-CoV-2 spike S1 and S2 domains and nucleocapsid (N) specific antibodies and were validated using sera from experimentally infected hamsters. We further validated our cELISA by comparing it with results obtained from the surrogate virus neutralization test (cPASS, GenScript) and indirect ELISA using anti-hamster horse radish peroxidase (HRP) conjugated reagents. Our initial cELISA was based on the ability of test antibodies to displace the binding of commercially obtained rabbit antibodies against viral proteins coated on the ELISA plate. Rabbit antibody reagents are expensive and anti-rabbit detection antibody may cross-react with other mammalian antibodies. Therefore, I explored the use of antibodies produced in hen eggs (IgY) as a substitute for rabbit sera. Hens were immunized against SARS-CoV-2 antigens: S1, S2 and N. IgY antibodies were purified from egg yolk, and the assay was optimized to use specific antibody and antigen combinations. Among S1, S2 and N-IgYs, only the S2-IgY based cELISA was specific and comparable with both the rabbit anti serum based cELISA and the surrogate virus neutralization test (cPASS). This assay will be a valuable tool which can be implemented in surveillance programs investigating exposure to and transmission of SARS-CoV-2 in multiple domestic, captive, or wildlife species.
SARS-CoV-2, Antibody, Competitive ELISA, IgY, Percent inhibition, Exposure
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