Clinical Utility of a Highly Sensitive Lateral Flow Immunoassay as determined by Titer Analysis for the Detection of anti-SARS-CoV-2 Antibodies at the Point-of-Care

  1. Amanda Haymond
  2. Claudius Mueller
  3. Hannah Steinberg
  4. K. Alex Hodge
  5. Caitlin Lehman
  6. Shih-Chao Lin
  7. Lucia Collini
  8. Heather Branscome
  9. Tuong Vi Nguyen
  10. Sally Rucker
  11. Lauren Panny
  12. Rafaela Flor
  13. Raouf Guirgus
  14. Richard Hoefer
  15. Giovanni Lorenzin
  16. Emanuel Petricoin
  17. Fatah Kashanchi
  18. Kylene Kehn-Hall
  19. Paolo Lanzafame
  20. Lance Liotta
  21. Alessandra Luchini

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Abstract

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.

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    SciScore for 10.1101/2020.07.30.20163824: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Binding was detected using 1:5,000 goat anti-human IgG-HRP conjugated secondary (Jackson Immunoresearch cat# 109-035-098) with TMB substrate (Fisher cat# 34028) for 12 minutes, with the reaction halted via 2 M sulfuric acid.
    anti-human IgG-HRP
    suggested: None
    Western Blot Analysis: Serum samples from patients 1F, 8F, 10F, and an uninfected negative control individual were examined for the presence of anti-ACE2 or anti-S antibodies.
    anti-ACE2
    suggested: None
    anti-S
    suggested: None
    Antibodies used for these experiments included α-Actin (Abcam # ab-49900).
    α-Actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    ELISA plates (Immulon 1B, ThermoFisher cat# 3355) were coated overnight at 4°C with 2 μg/mL SARS-CoV-2 spike RBD-mFc tag (Sino Biological cat# 40592-V05H) produced in HEK293 cells.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Vero cells were infected using the diluted sera and virus mixture and were incubated for 1h at 37°C.
    Vero
    suggested: None
    Whole cell extracts from A549 and Vero E6 were analyzed via BCA assay per the manufacturer’s recommendations (Thermo Fisher Scientific).
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Lateral Flow Immunoassay Titer Analysis: Combined anti-SARS-CoV-2 IgG/IgM lateral flow immunoassays were obtained from the following manufacturers: Pinnacle Biolabs SARS
    Pinnacle Biolabs
    suggested: None
    EasyDiagnosis Biomedicine Co., Ltd COVID-19 (SARS-CoV-2) IgM/IgG Antibody Test Kit, reference SA-2-D (EDiagnostics); SafeCare Bio-Tech COVID-19 IgG/IgM Rapid Test Device (WB/S/P) ref NCO-4022 (SafeCare), AcroBiotech 2019-nCoV IgG/IgM Rapid Test Cassette reference INCP-402 (AcroBiotech); LumiQuick Diagnostics Quick Profile 2019 nCoV IgG/IgM Test Card ref 71108B (LumiQuick); Cellex qSARS-COV-2 IgG/IgM Rapid Test (Cellex); CALTH Care Health AllCheck
    AcroBiotech
    suggested: None
    Krippendorff’s alpha statistic, and percent agreement as calculated by STATA 13 (StataCorp. 2013. Stata Statistical Software:
    STATA
    suggested: (Stata, RRID:SCR_012763)
    StataCorp
    suggested: (Stata, RRID:SCR_012763)
    To determine sensitivity and specificity of LFI tests in the healthcare setting for saliva and fingerstick blood, receiver-operator characteristic (ROC) curves were generated with GraphPad Prism version 8.4.3.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Two by two tables used to determine positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity were generated using Microsoft Excel.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    Results from OddPub: Thank you for sharing your data.

    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

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  2. Version published to 10.1101/2020.07.30.20163824v1 on medRxiv