Development and Validation of a Multiplex, Bead-based Assay to Detect Antibodies Directed Against SARS-CoV-2 Proteins

  1. Robert A. Bray
  2. Jar-How Lee
  3. Peter Brescia
  4. Deepali Kumar
  5. Thoa Nong
  6. Remi Shih
  7. E. Steve Woodle
  8. Jonathan S. Maltzman
  9. Howard M. Gebel

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Abstract

Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining the status of antibodies against SARS-CoV-2 in both candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 in this population. While there are multiple tests to detect antibodies to SARS-CoV-2, their performance is variable. Tests vary according to their platforms and the antigenic targets which make interpretation of the results challenging. Furthermore, for some assays, sensitivity and specificity are less than optimal. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses rather than SARS-CoV-2.

Methods.

This study describes the development and validation of a high-throughput multiplex antibody detection assay.

Results.

The multiplex assay has the capacity to identify, simultaneously, patient responses to 5 SARS-CoV-2 proteins, namely, the full spike protein, 3 individual domains of the spike protein (S1, S2, and receptor binding domain), and the nucleocapsid protein. The antibody response to the above proteins are SARS-CoV-2-specific, as antibodies against 4 common coronaviruses do not cross-react.

Conclusions.

This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting. Test configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.02.20185199: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationInitially, 96 serum samples from a pre-COVID-19 time period (October 2018) were randomly selected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The polyclonal antibodies used for bead validation were: SARS-CoV-2 NC Protein (Cat.#: 40588-T62, 1:1000 dilution), SARS-CoV-2 Spike (Cat#: 40589-T62; 1:1000 dilution), SARS-CoV-S2 (Cat#: 40590-T62; 1:1000 dilution), SARS-CoV S1 (Cat#: 40150-T62, 1:1000 dilution), SARS-CoV NC Protein (Cat#: 40143-T62, 1:1000 dilution), MERS-S1 (Cat#: 40069-T52, 1:1000
    SARS-CoV-S2
    suggested: None
    MERS-S1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Additionally, the kit incorporates Spike S1 fragments from six other coronaviruses, namely HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43, MERS-CoV and SARS-CoV.
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Software and Algorithms
    SentencesResources
    Final analysis of MFI (Mean Fluorescence Intensity) was performed using Microsoft Excel.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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:
    An additional important limitation of such single target assays is that they fail to assess the breadth of a patient’s response. In fact, recent studies reveal that patient antibody responses to SARS CoV-2 are not uniform. For example, while convalescent plasma donors and patients who have recovered from SARS-CoV-2 infection have detectable RBD antibodies that are neutralizing, RBD antibodies detected in pediatric COVID-19 patients who developed multisystem inflammatory syndrome are not neutralizing (11). The collective published data illustrate the gap in our understanding the complexities of the humoral immune response to COVID-19. Assays that interrogate a response to a single viral antigen limit our ability to understand fully the immune responsiveness to SARS-CoV-2. This is evidenced by the response of subject #8 who showed strong reactivity against the Full Spike protein but was negative for S1, S2 and RBD. The low level of NC protein reactivity also did not exceed the 3 SD cutoff to consider the reaction as positive. Since S1, S2 and RBD proteins are elements of the Full Spike protein; this reactivity could reflect responsiveness to a unique cross-reactive epitope not related to SARS-CoV-2 infection. This reactivity pattern was not observed in any other SARS-CoV-2 negative sample either pre- or post-COVID-19. Furthermore, the reactivity pattern of subject #8 extended well back into the global pre-COVID-19 era. The exact nature of this reactivity is under further invest...

    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.

    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 10.1097/tp.0000000000003524
  3. Version published to 10.1101/2020.09.02.20185199v1 on medRxiv