The Theory and Practice of the viral dose in neutralization assay: insights on SARS-CoV-2 “doublethink” effect

  1. Alessandro Manenti
  2. Eleonora Molesti
  3. Marta Maggetti
  4. Alessandro Torelli
  5. Giulia Lapini
  6. Emanuele Montomoli

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Abstract

Due to the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for reliable high-throughput serological assays in order to evaluate the immunological responses against SARS-COV-2 virus and to enable population screening, as well as vaccines and drug’s efficacy testing. Several serological assays for SARS-CoV-2 are now becoming available in the market. However, it has also become extremely important to have well-established assays with desirable high sensitivity and specificity. To date, the micro-neutralization (MN) assay, is currently considered the gold-standard being capable of evaluating and detecting, functional neutralizing antibodies (nAbs). Several protocols exist for microneutralization assays which vary in several steps of the protocol: cell seeding conditions, number of cells seeded, virus amount used in the infection step, virus-serum-cells incubation period etc. These potential differences account for a high degree of variability and inconsistency of the results and using a harmonized protocol for the micro-neutralization assay could potentially solve this.

Given this situation, the main aim of our study was to carry out SARS-CoV-2 wild type virus MN assay in order to investigate which optimal tissue culture infective dose 50 (TCID 50 ) infective dose in use is the most adequate choice for implementation in terms of reproducibility, standardization possibilities and comparability of results. Therefore, we assessed the MN by using two different viral infective doses: a standard dose of 100 TCID 50 /well and a lower dose of 25 TCID 50 /well. The results obtained, yielded by MN on using the lower infective dose (25 TCID 50 ), were in line with those obtained with the standard infective dose; in some cases, however, we detected a titre that was one or two dilution steps higher, which maintained all negative samples negative. This suggesting that the lower dose can potentially have a positive impact on the detection and estimation of neutralizing antibodies present in a given sample, showing higher sensitivity but similar specificity and therefore, it would require a more accurate assessment and cross-laboratories standardisation especially when MN is employed as serological assay of choice for pre-clinical and clinical studies.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.16.342428: (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
    (ELISA): Specific anti-SARS-CoV-2 IgG antibodies were detected through a commercial ELISA kit (Euroimmun, Lübeck, Germany).
    anti-SARS-CoV-2 IgG
    suggested: None
    According to the manufacturer, cross-reactions may occur with anti-SARS-CoV(−1) IgG antibodies, due to the close relationship between SARS-CoV(−1) and SARS-CoV-2, while cross-reactions with other human pathogenic CoVs (MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43) are excluded.
    anti-SARS-CoV(−1) IgG
    suggested: None
    SARS-CoV-2
    suggested: None
    HCoV-HKU1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The virus was titrated in serial 1 log dilutions (from 1 log to 11 log) to obtain a 50% tissue culture infective dose (TCID50) on 96-well culture plates of VERO and VERO E6 cells.
    VERO E6
    suggested: RRID:CVCL_XD71)
    According to the manufacturer, cross-reactions may occur with anti-SARS-CoV(−1) IgG antibodies, due to the close relationship between SARS-CoV(−1) and SARS-CoV-2, while cross-reactions with other human pathogenic CoVs (MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43) are excluded.
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Software and Algorithms
    SentencesResources
    Statistics analysis: Data analysis was performed using GraphPad Prism Version 5.
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.10.16.342428 on bioRxiv