A rapid bead-based assay for screening of SARS-CoV-2 neutralizing antibodies

  1. Santhik Subhasingh Lupitha
  2. Pramod Darvin
  3. Aneesh Chandrasekharan
  4. Shankara Narayanan Varadarajan
  5. Soumya Jaya Divakaran
  6. Sreekumar Easwaran
  7. Shijulal Nelson-Sathi
  8. Perunthottathu K Umasankar
  9. Sara Jones
  10. Iype Joseph
  11. Madhavan Radhakrishna Pillai
  12. Thankayyan Retnabai Santhoshkumar

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Abstract

Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost-effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. We describe a bead-based screening assay for S1-neutralization using recombinant fluorescent proteins of hACE2 and SARS-CoV2-S1, immobilized on solid beads employing nanobodies/metal-affinity tags. Nanobody-mediated capture of SARS-CoV-2-Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single-color fluorescent imaging of ACE2-GFPSpark binding to His-tagged S1-Receptor Binding Domain (RBD-His) immobilized beads. The second approach is dual-color imaging of soluble ACE2-GFPSpark to S1-Orange Fluorescent Protein (S1-OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening.

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  1. Version published to 10.1093/abt/tbac007
  2. ScreenIT

    SciScore for 10.1101/2021.10.13.464050: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells and expression vectors: HEK293T cells and SiHa cells were procured from the central cell line repository of RGCB (CCL
    SiHa
    suggested: KCB Cat# KCB 2013026YJ, RRID:CVCL_0032)
    CaCo-2 cells were obtained from ATCC.
    CaCo-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    DLD-1 cells were obtained from Sigma (Merck #90102540).
    DLD-1
    suggested: ECACC Cat# 90102540, RRID:CVCL_0248)
    HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).
    HEK293T
    suggested: None
    The mixture was added to the HEK293T-HA-ACE2 cells for 4 hours, followed by fresh medium, and wells with IgG served as control.
    HEK293T-HA-ACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse immunization and serum preparation: BALB/c mice were immunized with purified RBD-His protein expressed in E. coli as per the standard protocol approved by IAEC.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    ) Spike S1 Gene with C terminal OFP Spark fluorescent tag (pCMV3-C-OFPSpark) was obtained from Sino biological (#VG40591-ACR)
    pCMV3-C-OFPSpark
    suggested: None
    Full-length DNA clone of Human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 with C terminal GFPSpark tag (pCMV3-hACE2-GFPSpark) was obtained from Sino biological (#HG10108-ACG).
    pCMV3-hACE2-GFPSpark
    suggested: None
    The mammalian expression vector for soluble ACE2 pcDNA3-sACE2 (WT)-sfGFP (#145171) was obtained from Addgene.
    pcDNA3-sACE2
    suggested: None
    The packaging vectors and Lenti ZsGreen were from Addgene (pHIV-Zsgreen #18121
    pHIV-Zsgreen
    suggested: RRID:Addgene_18121)
    Immobilization of GFP Nanobody to Agarose beads: The bacterial expression vector for anti-GFP nanobody pGEX-6P-1 was procured from Addgene.
    pGEX-6P-1
    suggested: None
    HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).
    psPAX2
    suggested: RRID:Addgene_12260)
    pCDNA 3.1_Spike_Del19
    suggested: None
    Software and Algorithms
    SentencesResources
    The images were acquired and analyzed using NIS-Elements software.
    NIS-Elements
    suggested: (NIS-Elements, RRID:SCR_014329)

    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 22. 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.10.13.464050v1 on bioRxiv