COVID-19 Serology in New York State Using a Multiplex Microsphere Immunoassay

  1. Danielle T. Hunt
  2. Jennifer L. Yates
  3. Karen E. Kulas
  4. Kyle Carson
  5. Theresa Lamson
  6. Valerie Demarest
  7. Andrea Furuya
  8. Kelly Howard
  9. Mary Marchewka
  10. Randy Stone
  11. Heidi Tucker
  12. Casey Warszycki
  13. Jim Yee
  14. He S. Yang
  15. Sabrina Racine-Brzostek
  16. Zhen Zhao
  17. Monir Ejemel
  18. Qi Li
  19. Yang Wang
  20. Sebastian Fernando
  21. Francesca La Carpia
  22. Eldad A. Hod
  23. Kathleen A. McDonough
  24. William T. Lee

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Abstract

The emergence of SARS-CoV-2, leading to COVID-19, necessitated the development of new molecular and serological tests. Here, we describe a multiplexed serological assay developed as the global pandemic moved into New York State in the spring of 2020. The original microsphere immunoassay used a target antigen from the SARS-CoV-1 virus responsible for the 2003 SARS outbreak, but evolved to incorporate multiple SARS-CoV-2 protein antigens (nucleocapsid, spike and spike domains, spike and nucleocapsid proteins from seasonal human coronaviruses). Besides being highly versatile due to multiplex capabilities, the assay was highly specific and sensitive and adaptable to measuring both total antibodies and antibody isotypes. While determining the assay performance characteristics, we were able to identify antibody production patterns (e.g., kinetics of isotypes, individual variations) for total antibodies and individual antibody classes. Overall, the results provide insights into the laboratory response to new serology needs, and how the evolution and fine-tuning of a serology assay helped contribute to a better understanding of the antibody response to SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.12.21257125: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: COVID-19 Serum Samples: Studies were performed on sera from deidentified specimens submitted to the New York State Department of Health in response to a broad request for assay development and validations.
    IRB: All testing and archiving of human specimens were done under a declared New York State Public Health Emergency and also approved by NYSDOH Institutional Review Board (IRB 20-021).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Serum samples (25 μL at 1:100 dilution) and antigen-conjugated microspheres (25 μL at 5×104 microspheres/mL) were mixed and incubated 30 minutes at 37°C before washing and further incubation with phycoerythrin (PE)-conjugated secondary antibody.
    antigen-conjugated microspheres ( 25
    suggested: None
    The PE-conjugated antibodies were chosen to specifically recognize, as indicated, total antibodies (pan-Ig), or, individually IgM, IgA, IgG, IgG1, IgG2, IgG3, IgG4.
    IgG1
    suggested: None
    IgG4
    suggested: None
    The target antigen is the SARS-CoV-2 N protein and only IgG antibodies were measured.
    only IgG antibodies
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For production of the recombinant SARS-CoV-1 Nucleocapsid protein, a cDNA copy of the N gene of SARS-CoV was produced by reverse transcription-PCR of RNA purified from Vero E6 cells that had been infected with SARS-CoV (strain Urbani; GenBank accession number AY278741).
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    The synthetic gene encoding the Receptor Binding Domain (RBD) a.a. 319-541 and S1 subunit a.a. 1-604 of the S glycoproteins were cloned into pcDNA 3.1 Myc/His in-frame with c-Myc and 6-histidine epitope tags that enabled detection and purification.
    pcDNA 3.1
    suggested: RRID:Addgene_20407)
    The N gene cDNA was ligated into the bacterial expression vector pET-28a(+) (Novagen (Merck-Millipore) Burlington,MA) for expression of carboxy-terminal His6-tagged full-length N protein.
    pET-28a(+
    suggested: RRID:Addgene_108949)
    Software and Algorithms
    SentencesResources
    4.3 Reagents: For MIAs, wash buffer and phosphate buffered saline pH 7.4, 0.05% sodium azide (PBS-TN) were purchased from Sigma (Sigma Aldrich, St. Louis, MO).
    Sigma ( Sigma Aldrich
    suggested: None
    ) Vitros anti-SARS-CoV-2 IgG chemiluminescent immunoassay; 2) the Abbott Laboratories (Abbott Park, IL)
    Abbott Laboratories
    suggested: None
    The Abbott Laboratories SARS-CoV-2 IgG assay is a qualitative chemiluminescence test that we performed on the Abbott Architect i1000SR automated immunoassay analyzer.
    Abbott Architect
    suggested: (Abbott ARCHITECT i1000sr System, RRID:SCR_019328)
    0%); Specificity, 99.9% (99.4% - 100%). 4.7 Reporting of Statistical Methods: All graphs and statistical analyses to determine Spearman’s rank correlation coefficients were done using Prism 9.0 (Graphpad, San Diego, CA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    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: 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.
    • Thank you for including a protocol registration statement.

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

    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 10.1101/2021.05.12.21257125v1 on medRxiv