A simple protein-based surrogate neutralization assay for SARS-CoV-2

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  1. SciScore for 10.1101/2020.07.10.197913: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study approval: All samples were collected after Research Ethics Board (REB) review.
    Consent: All participants have provided informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    For the CR3022 antibody, the harvested medium was incubated with rProtein A Sepharose FF resin (GE healthcare).
    suggested: None
    Commercial antibodies tested also included a human IgG chimeric antibody from GenScript (SARS-CoV-2 spike S1 Antibody (HC2001), GenScript #A02038) and two SARS-CoV-2 spike Antibodies from Active Motif (AM002414, #91349; AM001414, #91361)
    suggested: None
    suggested: (Proteintech Cat# 91349-PTG, RRID:AB_2882951)
    Direct ELISA assay for the identification of antibodies to the RBD: For the manual single point ELISAs in 96-well format, concentrations and incubation times were optimized to maximize the separation between anti-RBD levels in convalescent plasma or serum from that of pre-COVID era banked serum while maintaining the required levels of antigens as low as possible.
    suggested: None
    A chimeric human anti-spike antibody (SARS-CoV-2 spike S1 Antibody (HC2001), Genscript #A02038) was added to a set of wells on each plate as a serial dilution (1:5,000 to 1:80,000 or 10 ng to 0.63 ng per well in four steps) to enable cross-plate comparisons.
    suggested: None
    Experimental Models: Cell Lines
    A cDNA encoding VHH72 fused to an ADCC-attenuated human IgG1 Fc domain (hFc1X7, from patent US 2019 352 383A1) was codon-optimized for expression in Cricetulus griseus (CHO cells), synthesized by GenScript and cloned into the pTT5™ plasmid (33).
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    The pTT5-VHH72hFc1X7 plasmid was transiently expressed in CHO55E1 cells (34) using PEI-Max transfection reagent (Polysciences, Warrington, PA) and a slightly modified protocol as described previously (35).
    suggested: None
    Patient sera and SARS-CoV-2 (USA/WA-1/2020, BEI Resources, #NR-52281) were diluted in Vero E6 cell culture maintenance medium (Eagle’s Minimal Essential Medium, 2% heat-inactivated fetal bovine serum, 200 U/ml Penicillin G, 200 U/ml Streptomycin).
    Vero E6
    suggested: None
    HEK293T cells were transduced with ACE2 lentivirus at an MOI <1 and selected with puromycin (1 μg/mL) to generate a stable population.
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    The virus titers were evaluated using HEK293T-ACE2/TMPRSS2 cells at 10K cells per well on a Poly-L-Lysine [5-10 μg/mL] coated 96-well plate using HI10 media (10% heat-inactivated FBS, 1% Pen/Strep) and a virus dilution resulting in >1000 relative luciferase units (RLU) over control (~1:100 virus stock dilution).
    suggested: None
    For the neutralization assay, 2.5-fold serial dilutions of the serum samples were incubated with diluted virus at a 1:1 ratio for 1 hr at 37 °C before being transferred to plated HEK293-ACE2/TMPRSS2 cells and incubated for an additional 48 hr at 37 °C and 5% CO2.
    suggested: None
    Software and Algorithms
    For the lentiviral pseudotyping assays, 50% inhibitory concentration or dilution (IC50 or ID50) were calculated with non-linear regression [log(inhibitor) vs. normalized response - Variable slope] using GraphPad Prism 8 (GraphPad Software Inc.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    There are limitations to the assay, however, that need to be acknowledged. First, the snELISA is limited to the detection of neutralizing antibodies that function by blocking the interaction between the RBD and ACE2. While by no means dominant, examples of antibodies that neutralize by other mechanisms are beginning to emerge (21-23). The snELISA, in conjunction with a neutralization assay, could be used to identify further such examples. As with those identified in this work, the outliers (e.g. those with high viral neutralization titers but low snELISA levels) provide a starting point for further work aimed at understanding the mechanisms of antibody-mediated neutralization. Another limitation of our approach is that the current assay cannot directly map the epitopes targeted by the various antibodies. Undoubtedly, the antibodies detected by the snELISA bind to different sites on the RBD, a suggestion supported by the structures of neutralizing antibody Fabs in complex with the SARS-CoV-2 RBD. In one example, two different neutralizing antibodies that bind to different epitopes on the RBD were found to synergistically mediate viral neutralization (24). While in the current study we simply wanted to provide evidence of antibodies that could block the RBD-ACE2 interaction, the snELISA could be adapted to provide information on the site of antibody binding. As recently shown, a series of structure-guided point mutants in the RBD could be used to infer where on the RBD the anti...

    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

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