Analytical and diagnostic performances of a high-throughput immunoassay for SARS-CoV-2 IgM and IgG

  1. Andrea Padoan
  2. Chiara Cosma
  3. Paolo Zaupa
  4. Mario Plebani

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Abstract

Reliable SARS-CoV-2 serological assays are required for diagnosing infections, for the serosurveillance of past exposures and for assessing the response to future vaccines. In this study, the analytical and clinical performances of a chemiluminescent immunoassays for SARS-CoV-2 IgM and IgG detection (Mindray CL-1200i), targeting Nucleocapsid (N) and receptor binding domain (RBD) portion of the Spike protein, were evaluated.

Methods

Precision and linearity were evaluated using standardized procedures. A total of 157 leftover serum samples from 81 hospitalized confirmed COVID-19 patients (38 with moderate and 43 with severe disease) and 76 SARS-CoV-2 negative subjects (44 healthcare workers, 20 individuals with rheumatic disorders, 12 pregnant women) were included in the study. In an additional series of 44 SARS-CoV-2 positive, IgM and IgG time kinetics were also evaluated in a time-period of 38 days.

Results

Precision was below or equal to 4% for both IgM and IgG, in all the studied levels, whilst a slightly significant deviation from linearity was observed for both assays in the range of values covering the manufacturer’s cut-off. Considering a time frame ≥ 12 days post symptom onset, sensitivity and specificity for IgM were 92.3% (95%CI:79.1%-98.4%) and 92.1% (95%CI:83.6%-97.0%). In the same time frame, sensitivity and specificity for IgG were 100% (95%CI:91.0%-100%) and 93.4% (95%CI:85.3%-97.8%). The assays agreement was 73.9% (Cohen’s kappa of 0.373). Time kinetics showed a substantial overlapping of IgM and IgG response, the latter values being elevated up to 38 days from symptoms onset.

Conclusions

Analytical imprecision is satisfactory as well as the linearity, particularly when taking into account the fact that both assays are claimed to be qualitative. Diagnostic sensitivity of IgG was excellent, especially considering specimens collected ≥12 days post symptom onset. Time kinetics suggest that IgM and IgG are detectable early in the course of infection, but the role of SARS-CoV-2 antibodies in clinical practice still requires further evaluations.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.11.20.20235267: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: 2.7 Ethical statement: The study protocol (number 23307) was approved by the Ethics Committee of the University-Hospital of Padova.
    Consent: All the patients who agreed to participate provided written consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variable2.4 Samples included in the study: A total of 157 leftover serum samples from 81 hospitalized COVID-19 patients (38 classified with moderate and 43 with severe disease according to the WHO interim guidance) and 76 SARS-CoV-2 negative subjects [44 healthcare workers (NHW), 20 patients with rheumatic disorders (RD), 12 pregnant women (Pr)] were included in the study (Table 1).

    Table 2: Resources

    Antibodies
    SentencesResources
    2.1 Analytical methods: The CL-series SARS-CoV-2 IgG and IgM assays are a two-step chemiluminescent immunoassays for detection of IgG and IgM SARS-CoV-2 antibodies in human serum or plasma (or EDTA or heparin), analyzed on the fully automated platform Mindray
    SARS-CoV-2 IgG
    suggested: None
    IgM SARS-CoV-2
    suggested: None
    Alkaline phosphatase-labeled anti-human IgG or IgM monoclonal antibodies are added to the reaction to form sandwich structure with microparticles captured anti-SARS-CoV-2 antibodies.
    anti-human IgG
    suggested: None
    IgM
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    In brief, two serum pools with a measured IgM and IgG antibody values of 1.4 kAU/L and 18.28 kAU/L (high-level pools), respectively, were serially diluted with a low antibody values serum pools (0.05 kAU/L for IgM and 0.01 kAU/L IgG).
    IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    The GraphPad Prism version 9.0 for Windows was used to evaluate kinetic data.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Stata v16.0 (Statacorp, Lakeway Drive, TX, USA) was used as statistical software, while the “diagt” module was used to estimate diagnostic performances.
    Statacorp
    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: We detected the following sentences addressing limitations in the study:
    This study presents several limitations. First, only Mod/Sev SARS-CoV-2 individuals were included in the study, as sera from asymptomatic/paucisymptomatic subjects were not available. Second, IgM and IgG kinetics has been assessed in a limited period, while a longer monitoring should be necessary in order to estimate the entire trend of humoral immune response to COVID-19 infection, particularly the eventual antibodies decay. Third, comorbidities were not evaluated since not completely recorded. In conclusion, Mindray CL-1200i SARS-CoV-2 IgM and IgG antibodies assays show excellent precision results and linearity, comparable to that claimed by manufacturer’s insert. Clinical performances in terms of both AUROC and sensitivities were also very elevated, especially after 12 days post symptom onset. The presence of a limited number of false positive results prevented to achieve 100% specificity, thus indicating that this point could be further improved. Finally, the overlapping time-kinetics between IgM and IgG specific antibodies, particularly in the early stages of the infection, suggests that the diagnostic value of IgM assay is still debatable.

    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 10.1101/2020.11.20.20235267v1 on medRxiv