Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2

  1. Nadezhda V. Antipova
  2. Tatyana D. Larionova
  3. Andrei E. Siniavin
  4. Maria A. Nikiforova
  5. Vladimir A. Gushchin
  6. Igor I. Babichenko
  7. Alexey V. Volkov
  8. Michail I. Shakhparonov
  9. Marat S. Pavlyukov

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Abstract

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients’ tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.

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  1. Version published to 10.3390/ijms21239167
  2. ScreenIT

    SciScore for 10.1101/2020.08.29.272963: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experiments were carried out under an Institutional Animal Care and Use Committee (IACUC) approved protocol according to NIH guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing with TBST wells were incubated with HRP-conjugated anti-mouse secondary antibodies (Thermo Fisher) (1:10000 dilution in TBST) and developed with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher) according to the manufacturer’s protocol.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell Culture: HEK293 and HT1080 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, 1mM Na-pyruvate and penicillin-streptomycin mixture (100μg/ml).
    HT1080
    suggested: None
    Deglycosylation: Deglycosylation of S protein from the lysate of HEK293 cells transfected with pTwist-EF1a-nCoV-2019-S-2xStrep plasmid was performed as described previously [35] using PNGase F (New England Biolabs).
    HEK293
    suggested: None
    Generation of SARS-CoV2 pseudovirus particles: HEK293FT cells in T75 flask were cotransfected with 6 μg of pTwist-EF1a-nCoV-2019-S-2xStrep plasmid, 9 μg of psPAX2 plasmid and 12 μg of pCDH-GFP-IRES-Puro plasmid [36].
    HEK293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice immunization and hybridoma fusion: 5 weeks old BALB/c mice were immunized according to the standard protocol [32] with several modifications.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    Immunoglobulin sequences were analyzed using IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE) and SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/) software.
    IgBLAST
    suggested: (IgBLAST, RRID:SCR_002873)
    BLASTn
    suggested: (BLASTN, RRID:SCR_001598)
    https://blast.ncbi.nlm.nih.gov/Blast.cgi
    suggested: (TBLASTX, RRID:SCR_011823)
    http://www.cbs.dtu.dk/services/SignalP/
    suggested: (SignalP, RRID:SCR_015644)

    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.
    • 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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  3. Version published to 10.1101/2020.08.29.272963v1 on bioRxiv