Diagnostic TR-FRET assays for detection of antibodies in patient samples

  1. Hong Yue
  2. Radosław P. Nowak
  3. Daan Overwijn
  4. N. Connor Payne
  5. Stephanie Fischinger
  6. Caroline Atyeo
  7. Evan C. Lam
  8. Kerri St. Denis
  9. Lauren K. Brais
  10. Yoshinobu Konishi
  11. Romanos Sklavenitis-Pistofidis
  12. Lindsey R. Baden
  13. Eric J. Nilles
  14. Elizabeth W. Karlson
  15. Xu G. Yu
  16. Jonathan Z. Li
  17. Ann E. Woolley
  18. Irene M. Ghobrial
  19. Jeffrey A. Meyerhardt
  20. Alejandro B. Balazs
  21. Galit Alter
  22. Ralph Mazitschek
  23. Eric S. Fischer

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Abstract

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  1. Version published to 10.1016/j.crmeth.2023.100421
  2. ScreenIT

    SciScore for 10.1101/2020.09.01.20184101: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All samples were collected after subjects provided signed informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The concentration of protein conjugate, Cab (M) was determined using equation 2:

    where ε is the antibody extinction coefficient at A280, equal to 210,000 M-1cm-1 for IgG class anti IgG/IgM/IgA Ab, 240,000 M-1cm-1 for S protein, 80,200 M-1cm-1 for RBD and b is path length in cm (0.1 cm).

    anti IgG/IgM/IgA
    suggested: None
    The degree of labeling (DOL) was calculated using equation 4: TR-FRET assay for RBD: Titration of CR3022 IgG/IgM/IgA1 antibody or dilution of tested human serum samples was added to assay mix with final concentrations of 15 nM Tb-labeled RBD, 250 nM BODIPY-labeled αIgG/αIgM/αIgA in a buffer containing PBS, 0.05% Tween-20
    CR3022 IgG/IgM/IgA1
    suggested: None
    50 μl/well of diluted detection antibody solution (HRP-anti human IgG Bethyl Laboratory #A80-104P) was added to the wells and incubated for 30 minutes at room temperature.
    HRP-anti human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Antibodies were expressed in Expi293T cells following manufacturer protocol (Thermo Fischer Scientific, A14525) with transfection ratios of 1:1 or 2:1 of heavy to light chain.
    Expi293T
    suggested: None
    Software and Algorithms
    SentencesResources
    All samples were tested on an in house validated RBD-specific ELISA as well as on FDA approved Roche and Abbott assays that test for antibodies to N protein.
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    50μl/well of diluted detection antibody solution (HRP-anti human IgG, IgA or IgM; Bethyl Laboratory #A80-104P, A80-100P, A80-102P) was added to the wells and incubated for 30 minutes at room temperature.
    Bethyl Laboratory
    suggested: (Bethyl, RRID:SCR_013554)
    Statistics: Statistical calculations were performed using Prism 8.0.2 and R v3.6.1; packages ggplot2.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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.
    • 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.

  3. Rapid Reviews Infectious Diseases

    Thomas Moran, J. Andrew Duty, Thomas Kraus

    Review 2: "Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma"

    This pre-print offers a FRET-based diagnostic platform that is faster and less labor-intensive than ELISA-based diagnostics. The claims should be considered reliable.

  4. Rapid Reviews Infectious Diseases

    Xi Chen, Simin Xia

    Review 1: "Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma"

    This pre-print offers a FRET-based diagnostic platform that is faster and less labor-intensive than ELISA-based diagnostics. The claims should be considered reliable.

  6. Version published to 10.1101/2020.09.01.20184101v1 on medRxiv