A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2

  1. Xiaojian Han
  2. Yingming Wang
  3. Shenglong Li
  4. Chao Hu
  5. Tingting Li
  6. Chenjian Gu
  7. Kai Wang
  8. Meiying Shen
  9. Jianwei Wang
  10. Jie Hu
  11. Ruixin Wu
  12. Song Mu
  13. Fang Gong
  14. Qian Chen
  15. Fengxia Gao
  16. Jingjing Huang
  17. Yingyi Long
  18. Feiyang Luo
  19. Shuyi Song
  20. Shunhua Long
  21. Yanan Hao
  22. Luo Li
  23. Yang Wu
  24. Wei Xu
  25. Xia Cai
  26. Qingzhu Gao
  27. Guiji Zhang
  28. Changlong He
  29. Kun Deng
  30. Li Du
  31. Yaru Nai
  32. Wang Wang
  33. Youhua Xie
  34. Di Qu
  35. Ailong Huang
  36. Ni Tang
  37. Aishun Jin

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Abstract

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC 50 ) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

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  1. Version published to 10.3389/fimmu.2021.653189
  2. Version published to 10.21203/rs.3.rs-87599/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2020.08.19.253369: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics Statement: The project “The application of antibody tests patients infected with SARS-CoV-2” was approved by the ethics committee of ChongQing Medical University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Then these PBMCs was incubated with mixed antibodies cocktail at 4 °C for 30 min (the antibodies cocktail including FITC-anti-human CD19 antibody (Biolegend, clone: SJ25C1)
    CD19
    suggested: None
    , BV421-anti-human IgD antibody (Biolegend, clone: IA6-2), PerCP-Cy5.5-anti-human IgG antibody (Biolegend, clone: M1310G05),
    BV421-anti-human IgD
    suggested: None
    PerCP-Cy5.5-anti-human IgG
    suggested: None
    The second PCR products were further cloned into the antibody linear expression cassettes or expression vectors to express full IgG1 antibodies.
    full IgG1
    suggested: None
    Plates were washed with phosphate-buffered saline, 0.05% Tween-20 (PBST) and ALP-conjugated goat anti-human IgG (H+L) antibody (Thermo Fisher) was added into each well and incubated at 37°C for 1 hour.
    anti-human IgG
    suggested: None
    Then ALP-conjugated anti-mouse-Ig-Fc antibody was added into the wells and incubated at 37°C for 30 min.
    anti-mouse-Ig-Fc
    suggested: None
    The CM5 chip (GE Healthcare) was coupled with an anti-human IgG-Fc antibody to capture 9000 response units antibodies.
    anti-human IgG-Fc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    After measuring the nucleic acid concentration, purified overlapping PCR products of paired heavy and light chain expression cassettes were co-transfected in HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    After incubation, the mixtures were then transferred into 96-well plates, which were seeded with Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Data analysis was performed utilizing the FlowJo software (FlowJo, LLC).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Half-maximal inhibitory concentrations (IC50) were calculated using the four-parameter logistic regression in GraphPad Prism 8.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Sequence analysis of antigen-specific mAb sequences: IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) and IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), MIXCR (https://mixcr.readthedocs.io/en/master/) and VDJtools (https://vdjtools-doc.readthedocs.io/en/master/overlap.html) tools were used to do the VDJ analysis and annotation, germline divergence for each antibody clone.
    IgBLAST
    suggested: (IgBLAST, RRID:SCR_002873)
    Abs DNA sequences were compared with each other by ClustalW (pairwise alignments) to analyze sequence similarity, and EvolView (https://www.evolgenius.info/evolview/) was used for the decoration of Phylogeny tree.
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    R packages (ggplot2, pheatmap) were used for the bar chart, heatmap and Cicos plot.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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
    NCT04497987RecruitingA Study of LY3819253 (LY-CoV555) and LY3832479 (LY-CoV016) i…
    NCT04426695RecruitingSafety, Tolerability, and Efficacy of Anti-Spike (S) SARS-Co…
    NCT04425629RecruitingSafety, Tolerability, and Efficacy of Anti-Spike (S) SARS-Co…

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  4. Version published to 10.1101/2020.08.19.253369v2 on bioRxiv
  5. Version published to 10.1101/2020.08.19.253369v1 on bioRxiv