Multi-species ELISA for the detection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic with millions of humans infected and hundreds of thousands of deaths. As the new disease - called COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers have been reported in dogs, species of felids and farmed mink. Other species have been shown to be sensitive under experimental conditions.

The extent of natural animal infections is however still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems have been evaluated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor binding domain (RBD) of SARS-CoV-2.

The newly established ELISA was evaluated using 59 sera from infected or vaccinated animals, including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and one cat, and a total of 220 negative sera. into antibodies from the same animal species.

Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% was achieved, and functionality with all species included in this study could be demonstrated. Therefore, a versatile and reliable ELISA protocol has been established that allows high throughput antibody detection in a wide range of animal species, which can be used for outbreak investigations, to assess seroprevalence in susceptible species or to screen for reservoirs or intermediate hosts.

Cells were transfected using the ExpiFectamine293 transfection kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Five to six days after transfection, supernatants were harvested by centrifugation at 6000xg for 20 min at 4°C.

Biotin was blocked by adding BioLock (IBA Lifesciences) as recommended, and the supernatant was purified using Strep-Tactin XT Superflow High Capacity Resin (iba lifesciences) according to the manufacturer's instructions. Finally, proteins were eluted with 50 mM biotin (in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA; pH 8.0).

Protein expression was verified by SDS-PAGE and Western blot using an anti-Strep-tag antibody conjugated to HRP (1/20,000 dilution; IBA lifesciences). Reactive bands were visualized using Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) and an Intas ChemoCam system (Intas Science Imaging Instruments GmbH).