Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals

  1. Kerstin Wernike
  2. Andrea Aebischer
  3. Anna Michelitsch
  4. Donata Hoffmann
  5. Conrad Freuling
  6. Anne Balkema-Buschmann
  7. Annika Graaf
  8. Thomas Müller
  9. Nikolaus Osterrieder
  10. Melanie Rissmann
  11. Dennis Rubbenstroth
  12. Jacob Schön
  13. Claudia Schulz
  14. Jakob Trimpert
  15. Lorenz Ulrich
  16. Asisa Volz
  17. Thomas Mettenleiter
  18. Martin Beer

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are validated 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 validated using 59 sera of infected or vaccinated animals including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% was achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.26.266825: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The expression of the proteins was verified by SDS-PAGE and western blotting using a HRP-conjugated anti-Strep-tag antibody (dilution 1/20,000, iba lifesciences).
    anti-Strep-tag
    suggested: None
    For chicken samples, an anti-chicken conjugate (Rabbit anti-Chicken IgY (H+L) Secondary Antibody, HRP; ThermoFisher Scientific) diluted 1/10,000 was applied.
    anti-Chicken IgY
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expi293 cells were grown in suspension in Expi293 expression medium (ThermoFisher Scientific) at 37 °C, 8 % CO2, and 125 rpm.
    Expi293
    suggested: RRID:CVCL_D615)
    Software and Algorithms
    SentencesResources
    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.
    BioLock
    suggested: None
    In addition, three sequential sera from a naturally SARS-CoV-2 infected cat were included (ProMED-mail, 2020).
    ProMED-mail
    suggested: (ProMed-Mail, RRID:SCR_010260)
    All analyses and visualizations were performed using GraphPad Prism version 8.0 for Windows (GraphPad Software, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.26.266825 on bioRxiv