Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG

  1. Veronica P Costantini
  2. Kenny Nguyen
  3. Zoe Lyski
  4. Shannon Novosad
  5. Ana C Bardossy
  6. Amanda K Lyons
  7. Paige Gable
  8. Preeta K Kutty
  9. Joseph D Lutgring
  10. Amanda Brunton
  11. Natalie J Thornburg
  12. Allison C Brown
  13. L Clifford McDonald
  14. William Messer
  15. Jan Vinjé

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Abstract

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

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  1. Version published to 10.4049/jimmunol.2100934
  2. ScreenIT

    SciScore for 10.1101/2021.09.03.21263078: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Specimen collection and processing: Saliva was collected at least 30 minutes after consumption of food or liquids.
    IRB: The study was approved by the Institutional Review Boards of the Oregon Health & Science University (IRB# 21230) (cohort II).
    Consent: Written informed consent was obtained from each participant.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Enzyme immunoassay (EIA) for detecting SARS-CoV-2-specific IgA and IgG in saliva: Convalescent sera from 3 SARS-CoV-2 patients with IgA and IgG antibodies against SARS-CoV-2 spike protein were used for the initial assay development.
    SARS-CoV-2 spike protein
    suggested: None
    Plates were incubated, washed, and bound antibodies were detected using HRP-conjugated goat anti-human IgA (Sera Care) diluted 1:4,000 in blocking buffer or anti-IgG (Sera Care)
    anti-human IgA
    suggested: None
    A standard curve for IgG was prepared by serial dilutions of purified human IgG (Sigma-Aldrich) and bound antibodies were detected using 1:16,000 diluted HRP-conjugated goat anti-human IgG (Sera Care).
    anti-human IgG
    suggested: None
    Sensitivity and specificity: The sensitivity (ability to identify samples with antibodies to SARS-CoV-2), and the specificity (ability to identify samples without antibodies to SARS-CoV-2) were defined as the values for which there is 95% probability that the estimated value can be obtained [17].
    SARS-CoV-2
    suggested: None
    To account for participant differences (e.g., severity of illness, immunocompetency, collection time, antibody secretion levels), SARS-CoV-2 specific IgA or IgG were normalized to 100 µg of total IgA or IgG, respectively.
    SARS-CoV-2 specific IgA or IgG
    suggested: None
    total IgA
    suggested: None
    When virus-specific antibodies could not be detected, but total antibodies were detected, SARS-CoV-2-specific IgA (or IgG) per 100 µg of total IgA (or IgG) were arbitrarily assigned as half the lower limit of detection, based on the standard curve of purified human IgA [18].
    SARS-CoV-2-specific IgA (or IgG
    suggested: None
    IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S) that was used in the assays was obtained from in suspension adapted HEK-293 cells as described previously [16].
    HEK-293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, California USA, www.graphpad.com), and p-values <0.05 were considered significant.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 several limitations. First, data on the onset and severity of disease was not always available. Second, collection of saliva samples did not always start on day 0 after onset of disease, not all participants provided samples at the same time points, and a limited number of longitudinal samples from asymptomatic individuals were available. Third, due to low sample volume, IgG testing could not be performed on all saliva samples. Fourth, saliva samples were not screened for the presence of antibodies against SARS-CoV-1, MERS-CoV or seasonal coronaviruses. We did test convalescent sera positive for seasonal coronavirus and showed no cross-reactivity. Others have shown some cross reactivity with SARS-CoV-1 and MERS-CoV which will be relevant only if these viruses circulate in the same population tested with our assay for antibodies against SARS-CoV-2. Finally, paired serum samples to correlate the immune response were not available; therefore, correlation between saliva and serum could not be evaluated. In summary, we developed and validated EIAs that are sensitive and specific to detect SARS-CoV-2 specific IgA and IgG antibodies in saliva. Significant fluctuations of salivary IgA and IgG antibodies levels were observed after infection. Detection of salivary antibodies may serve as an easy-to-employ screening method for population and transmission studies as well as evaluation of vaccine response, especially when collection of blood is challenging or not feasible.

    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.

    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.

  3. Version published to 10.1101/2021.09.03.21263078v1 on medRxiv