Development and performance evaluation of a rapid in-house ELISA for retrospective serosurveillance of SARS-CoV-2

  1. Bijon Kumar Sil
  2. Nowshin Jahan
  3. Md. Ahsanul Haq
  4. Mumtarin Jannat Oishee
  5. Tamanna Ali
  6. Shahad Saif Khandker
  7. Eiry Kobatake
  8. Masayasu Mie
  9. Mohib Ullah Khondoker
  10. Mohd. Raeed Jamiruddin
  11. Nihad Adnan

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Abstract

In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation.

Aim

To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients’ biological samples.

Methods

In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined.

Results

The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen’s kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases.

Conclusion

The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

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  1. Version published to 10.1371/journal.pone.0246346
  2. ScreenIT

    SciScore for 10.1101/2020.12.19.20248535: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethical Statement: This study was approved by National Research Ethics Committee (NREC) of Bangladesh.
    Consent: Well informed consents were obtained from all the participants.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For detection of immunocomplex of human IgG-capturing antigen commercially bought Goat anti-human IgG conjugated with HRP (horseradish peroxidase) (Native Antigen, UK) was utilized as secondary antibody. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Dojindo Molecular Technologies, USA) was used as HRP substrate (Wako, Japan) and the colour developed by TMB-peroxidase was stopped with 1.5 M H2SO4 (Sigma-Aldrich, Germany).
    human IgG-capturing
    suggested: None
    anti-human IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Statistical analysis was performed using STATA 13 (StataCorp, LP, College Station, Texas, USA) and the graphical presentation was made by using GraphPad Prism (7.05).
    STATA
    suggested: (Stata, RRID:SCR_012763)
    StataCorp
    suggested: (Stata, RRID:SCR_012763)
    GraphPad Prism
    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:
    There are certain limitations of our study that are to be addressed. Cross reactivity against SARS-CoV and other coronaviruses could not be assessed, and the size of sample collected within 14 days of symptom onset is relatively small. Nonetheless, the developed ELISA assays can be implemented in dengue-endemic countries due to their least cross reactivity and the assays are free from spectrum biasness. From our findings, we recommend the use of NCP and RBD Ig-ELISA kit for early detection studies of SARS-CoV-2 antibodies, with former being the better choice because of higher specificity. Whereas for retrospective sousveillance both S1- and RBD-ELISA can be implemented, with former being choice of assay for low prevalent areas. However, future endeavours should include longitudinal study with large cohort of population to access the antibody dynamics and persistence for the effective implementation of vaccine and eradication of COVID-19 from the population.

    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. Version published to 10.1101/2020.12.19.20248535v1 on medRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.12.10.20244350: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Three panels of serum samples comprising in total of 184 were collected from 134 individuals with their proper consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Analysis was performed with STATA 13 (
    STATA
    suggested: (Stata, RRID:SCR_012763)
    (StataCorp, LP, College Station, Texas, USA) and GraphPad Prism 7.05 were used for graphical presentation.
    StataCorp
    suggested: (Stata, RRID:SCR_012763)
    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:
    Certain limitations in our assay development exist that are to be addressed. Firstly, for cross reactivity test, no known respiratory sample was assessed, and secondly, the cohort sample size was actually inadequate to draw conclusions on samples collected within 0-14 days of symptom onset. In conclusion, this in-house ELISA demonstrates its usefulness for the early detection as well as for serosourveillence of SARS-CoV-2 IgG that developed against nucleocapsid proteins. This assay is easy to perform, cost-effective and results can be interpreted at 30 minutes of test run. Moreover, this test showed comparable level of performance against commercial FDA approved electrochemiluminescence immunoassay and detected IgG from SARS-CoV-2 infected patients. Hence, this SARS-CoV-2 NCP-IgG Rapid ELISA could be equally applied as one of the COVID-19 early sero-monitoring tools all over the COVID-19 pandemic countries.

    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.

  5. Version published to 10.1101/2020.12.10.20244350v1 on medRxiv