Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

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Abstract

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

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  1. SciScore for 10.1101/2021.03.05.434152: (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
    Virus and antibody-free cell culture medium served as a negative control.
    antibody-free cell culture medium
    suggested: None
    The diluted virus and diluted test samples were mixed 1:1 (vol/vol) and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 h to allow anti-SARS-CoV-2 antibodies to bind the virus.
    anti-SARS-CoV-2
    suggested: None
    Cells were stained with an anti-SARS antibody (SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit monoclonal antibody; Sino Biological #40143-R001), diluted to 0.125 μg/mL in 3% BSA/PBS blocking solution for 1 h at room temperature.
    anti-SARS
    suggested: None
    SARS-CoV/SARS-CoV-2 nucleocapsid antibody,
    suggested: (Thermo Fisher Scientific Cat# MA5-35943, RRID:AB_2866555)
    The cells were again washed three times with PBS and then stained with an Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific #A11037), diluted in BSA/PBS for 1 h in the dark at room temperature, and counterstained with Hoechst 33342 nuclear stain (Thermo Fisher Scientific #H3570).
    anti-rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# A-11037, RRID:AB_2534095)
    In the case of positive samples, S1-specific antibodies will bind to the viral antigens.
    S1-specific
    suggested: None
    To detect the bound antibodies, a second incubation is carried out using an enzyme-labelled antihuman IgG (peroxidase conjugate) catalyzing a color reaction.
    antihuman IgG
    suggested: (GeneTex Cat# GTX28798, RRID:AB_374523)
    Following a wash to remove any unbound enzyme-linked antibody, a substrate is added to the wells and color develops in proportion to the amount of IgG antibodies in the sample bound to the SARS-CoV-2 RBD antigen.
    SARS-CoV-2 RBD antigen.
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The virus was propagated at the Integrated Research Facility-Frederick in high containment (biosafety level 3 [BSL-3]) by inoculating Vero cells, acquired from the American Type Culture Collection (ATCC #CCL-81; Manassas, VA, USA).
    Vero
    suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
    #655948) containing Vero E6 cells and incubated at 37°C and 5% CO2 for 24 h.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The infected cells were incubated for 72 h in Dulbecco’s Modified Eagle Medium with L-glutamine (DMEM; Lonza, Walkersville, MD, USA) containing 2% heat-inactivated fetal bovine serum (FBS; SAFC Biosciences, Lenexa, KS, USA) in a humidified atmosphere at 37°C with 5% carbon dioxide (CO2).
    SAFC
    suggested: (SAFC, RRID:SCR_008554)
    A four-parameter logistical analysis was performed on the full dilution series using Prism (GraphPad Software, San Diego, CA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04344977CompletedCollection of Anti-SARS-CoV-2 Immune Plasma
    NCT04546581Active, not recruitingInpatient Treatment With Anti-Coronavirus Immunoglobulin (IT…


    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.