Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva

  1. Samantha H. Chiang
  2. Michael Tu
  3. Jordan Cheng
  4. Fang Wei
  5. Feng Li
  6. David Chia
  7. Omai Garner
  8. Sukantha Chandrasekaran
  9. Richard Bender
  10. Charles M. Strom
  11. David T. W. Wong

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Abstract

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H 2 O 2 /Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

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  1. Version published to 10.1371/journal.pone.0251342
  2. ScreenIT

    SciScore for 10.1101/2020.11.12.20230656: (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 variableBoth male and females, mostly non-smokers, 18-80 years of age, and a differing ethnicities were included.

    Table 2: Resources

    Antibodies
    SentencesResources
    The schematic of the Amperial™ SARS-CoV-2 IgG antibody is shown in Figure 1.
    SARS-CoV-2 IgG
    suggested: None
    Specific anti-S1 antibodies bind to the S1 antigen in the polymer.
    anti-S1
    suggested: None
    After rigorous washing procedures, the bound antibody is detected by using biotinylated anti-human IgG and then the signal is amplified by a standard streptavidin / horseradish peroxidase reaction that produces an electric current measured by the Amperial™ Reader in the nanoampere (nA) scale.
    nA
    suggested: None
    Biotinylated anti-human IgG secondary antibody (Thermofisher, Waltham, MA) at a stock concentration of 1.5 mg / mL was diluted 1:500 in Casein/PBS and 30 µL pipetted to the surface of each well and incubated for 10 minutes at room temperature followed by 6 wash cycles using PBS-T.
    anti-human IgG
    suggested: None
    Plasma SARS-CoV-2 ELISA Assay: We purchased FDA EUA ELISA kits EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgG antibodies (EUROIMMUN US, Mountain Lakes, NJ).
    Anti-SARS-CoV-2 ELISA Assay for detection of IgG
    suggested: None

    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.

    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. Version published to 10.1101/2020.11.12.20230656 on medRxiv