A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

  1. Juuso Rusanen
  2. Lauri Kareinen
  3. Lev Levanov
  4. Sointu Mero
  5. Sari H. Pakkanen
  6. Anu Kantele
  7. Fatima Amanat
  8. Florian Krammer
  9. Klaus Hedman
  10. Olli Vapalahti
  11. Jussi Hepojoki

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Abstract

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90–95% vs. 90–100%) and specificity (100% vs. 94–100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91–96% vs. 82–87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min “mix and read” assay, to detection of SARS-CoV-2 antibodies.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.01.20224113: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The data and samples were collected under research permit HUS/211/2020 and ethics committee approval HUS/853/2020 (Helsinki University Hospital, Finland).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For detection of anti-N antibodies, we found the optimal on-plate dilution for serum to be 1/25, and the optimal on-plate concentrations for AF-L and Eu-N to be 500 nM and 5 nM, respectively.
    anti-N
    suggested: None
    For detection of anti-S antibodies, we found an on-plate dilution of 1/100 for serum, AF-L concentration of 250 nM and Eu-S concentration of 5 nM optimal.
    anti-S
    suggested: None
    As secondary antibodies we made use of polyclonal rabbit anti-human IgA-horeseradish peroxidase (HRP), -IgM-HRP, and -IgG-HRP (all from Dako) at respective dilutions of 1:5000, 1:1500, and 1:6000.
    anti-human IgA-horeseradish peroxidase (HRP), -IgM-HRP,
    suggested: None
    -IgG-HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Next, we transfected adherent HEK293T cells with pCAGGS-SARS-CoV-S-Zeo plasmids using Fugene HD at 3.5:1 ratio, in suspension as described26.
    HEK293T
    suggested: None
    We adapted the HEK293T-spike-D5 cells for suspension culture by placing trypsinized cells into a spinner flask with Expi293 Expression Medium (ThermoFisher Scientific) with 100 µg/ml of Zeocin.
    HEK293T-spike-D5
    suggested: None
    Microneutralization: For the SARS-CoV-2 microneutralization assay we first cultured Vero E6 cells on 96-well plates (Thermo Scientific) overnight at +37°C in 2% MEM (Eagle Minimum Essential Media [Sigma-Aldrich] supplemented with 2% inactivated fetal bovine serum [Thermo Scientific], 2 mM L-glutamine [Thermo Scientific], 100 units penicillin, and 100 µg/ml streptomycin [Sigma-Aldrich]).
    Vero E6
    suggested: RRID:CVCL_XD71)

    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:
    Our study has some limitations. First, our SARS-CoV-2-positive samples originated from symptomatic patients. Individuals with asymptomatic infection may mount a significantly lower antibody response22, whereby the sensitivity of LFRET in such individuals might be lower. A second limitation is that antibodies against other coronaviruses, especially the widely circulating OC43, HKU1, NL63 and 229E, were not examined. Those antibodies potentially cross-reacting in the SARS-CoV-2 assay could reduce its specificity. However, the RT-PCR and neutralization results strongly indicate that the observed antibody responses were SARS-CoV-2-specific. In conclusion, this study demonstrates the applicability of the LFRET approach to detection of SARS-CoV-2 antibodies. While in sensitivity and specificity the assay appears to parallel ELISA, the new assay is as easy and rapid to perform as an LFA, requiring only combination of the diluted sample with a reagent mix and reading the result after 7 minutes. In prediction of neutralization capacity, the anti-S LFRET outperformed ELISA in specificity, at equal sensitivity.

    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 10.3390/v13020143
  3. Version published to 10.1101/2020.11.01.20224113v1 on medRxiv