Development of a rapid SARS-CoV-2 neutralisation test by detecting antibodies that block interaction of spike protein receptor-binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2)

  1. Ciaran Richardson
  2. Sarah Gildea
  3. Stephen Harkin
  4. Annie Gallagher
  5. Eimear Conroy
  6. Lorraine McClafferty
  7. Eibhlin McCole
  8. El Ouard Benchikh
  9. Cherith N. Reid
  10. Marshall Dunlop
  11. Sharon Savage
  12. Philip Lowry
  13. Jim Curry
  14. Robert I. McConnell
  15. John V. Lamont
  16. Stephen P. FitzGerald

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Abstract

The urgent need for a rapid and reliable Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralising antibody detection test compatible with routine clinical laboratory testing currently exists. This is necessary to provide accurate estimates of immunity and to monitor vaccine effectiveness. Utilising Biochip Array Technology (BAT), the Randox SARS-CoV-2 Biochip proxy virus neutralisation test (pVNT) has been developed. Immobilising SARS-CoV-2 Spike RBD on the Biochip surface, innovative assay design enables direct sample addition to the Biochip well without the need for off-board sample pre-incubation step. Results are reported within 1.5 hours and testing is carried out without the prerequisite of live virus or biosafety level 3 (BSL3) laboratory facilities. In this study, assay validation is performed using recombinant antibodies and clinical samples and an excellent correlation against conventional virus neutralisation methods is established (100% clinical specificity and 98% clinical sensitivity). Serial dilution of samples with high neutralising antibody levels demonstrate end-point sample dilutions comparable with those obtained using the SARS-CoV-2 microneutralisation test. Species independent neutralising antibody detection capacity of the SARS-CoV-2 Biochip pVNT is also demonstrated. The findings of this study exemplify the utility of the SARS-CoV-2 Biochip pVNT as a robust and reliable method for the accurate measurement of neutralising antibodies against SARS-CoV-2 and the availability of this test can now positively impact current testing deficiencies in this area.

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  1. ScreenIT

    SciScore for 10.1101/2021.06.03.21255116: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Immunisation and the collection of samples from sheep was carried out under Animals (Scientific Procedures) Act 1986 – Project License number PPL2797.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Human serum and plasma samples containing antibodies against endemic human coronaviruses (HKU1, OC43, 229E, NL63) and other non-coronavirus related pathogens were also collected.
    NL63
    suggested: None
    Recombinant antibodies: Humanised IgG recombinant antibodies SARS-CoV-1 RBD Antibody 1 (RAB0001), SARS-CoV-1 RBD Antibody 2 (RAB0003), SARS-CoV-2 RBD Antibody 1 (RAB0006) and SARS-CoV-2 RBD Antibody 2 (RAB0005) were supplied by Randox Laboratories Ltd.
    RAB0006
    suggested: None
    Sample characterisation using SARS-CoV-2 IgG (RBD & NP) assay: SARS-Cov-2 RBD IgG antibody levels were measured using the SARS-CoV-2 IgG (RBD & NP) array kit (Randox Laboratories Ltd, EV4447) as per the manufacturer’s instructions.
    SARS-CoV-2 IgG
    suggested: None
    NP
    suggested: None
    Samples with a % positivity (SP%) > 50% are reported as seropositive for SARS-CoV-2 RBD IgG antibodies.
    SARS-CoV-2 RBD IgG
    suggested: None
    SARS-CoV-2 neutralising antibodies if present, will bind RBD, inhibiting its ability to interact with ACE2 labelled horseradish peroxidase (ACE2 HRP).
    ACE2
    suggested: None
    ACE2 HRP
    suggested: None
    Samples with % inhibition > 50% are reported as seropositive for SARS-CoV-2 neutralising antibodies.
    SARS-CoV-2 neutralising antibodies.
    suggested: (Abcam Cat# ab273074, RRID:AB_2847846)
    Experimental Models: Cell Lines
    SentencesResources
    One-hour post-infection, the inoculum was replenished with fresh growth medium and plates were incubated overnight after which the Vero-E6 cells were fixed and immunostained to visualise infected cells.
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistics: The correlation between the SARS-CoV-2 Biochip pVNT and SARS-CoV-2 microneutralisation assay was calculated with Pearson’s correlation coefficients on MedCalc version 19.0.4.
    MedCalc
    suggested: (MedCalc, RRID:SCR_015044)

    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: We detected the following sentences addressing limitations in the study:
    This strategy, while worthwhile, has some limitations as such approaches cannot differentiate neutralising from non-neutralising antibodies and results obtained do not equate to protective immunity. Therefore, the SARS-CoV-2 Biochip pVNT test described in this study can positively impact current testing deficiencies in this area. ACE2 represents the primary receptor for viral entry into human cells and multiple studies have demonstrated that it is the RBD within the Spike protein of SARS-CoV-2 that interacts with ACE2 to precipitate viral entry15. During this study, we explored a pVNT format whereby ACE2 was immobilised on the Biochip surface and an alternative pVNT assay format whereby Spike RBD was immobilised on the Biochip surface. Good agreement in the detection of neutralising antibodies against SARS-CoV-2 was observed as demonstrated using recombinant neutralising antibodies and SARS-CoV-2 seropositive clinical samples. (Table 1A, 1B). The putative assay format whereby ACE2 is immobilized on Biochip surface requires a sample pre-incubation step where samples must be incubated off board with HRP labelled RBD prior to addition to ACE2 immobilized on the Biochip surface. This pre-incubation step is cumbersome with respect to manual immunoassay running, potentially increases the possibility of error due to increased immunoassay complexity and poses additional challenges with respect to achieving maximum throughput on automated immunoassay platforms. The alternative format ...

    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.

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

    About SciScore

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  2. Version published to 10.1101/2021.06.03.21255116v1 on medRxiv