Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

  1. Carolina de la Guardia
  2. Giselle Rangel
  3. Alcibiades Villarreal
  4. Amador Goodridge
  5. Patricia L. Fernández
  6. Ricardo Lleonart

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Abstract

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase–quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other ( P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

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  1. Version published to 10.1371/journal.pone.0257351
  2. ScreenIT

    SciScore for 10.1101/2021.04.30.21256406: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: Clinical serum samples from COVID-19 patients were collected as part of a cross-sectional study registered with the Panama Ministry of Health (No.1462) and was approved by the National Research Bioethics Committee (CNBI; No. EC-CNBI-2020-03-43).
    Consent: All the study participants were enrolled after informed consent was given.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISAs: The ELISA assays were implemented to detect SARS-CoV-2-specific IgG and IgM antibodies specific to S and RBD antigens.
    IgM
    suggested: None
    Specific IgG and IgM antibodies were detected with anti-human HRP conjugates: goat anti-human IgG (H+L), Thermo Fisher No. H10307, or goat anti-human IgM (Heavy chain), Thermo Fisher No. A18835, respectively.
    anti-human HRP
    suggested: (Thermo Fisher Scientific Cat# H10307, RRID:AB_2536551)
    anti-human IgG (H+L)
    suggested: (Thermo Fisher Scientific Cat# H10307, RRID:AB_2536551)
    anti-human IgM
    suggested: (Thermo Fisher Scientific Cat# A18835, RRID:AB_2535612)
    Software and Algorithms
    SentencesResources
    Specific IgG and IgM antibodies were detected with anti-human HRP conjugates: goat anti-human IgG (H+L), Thermo Fisher No. H10307, or goat anti-human IgM (Heavy chain), Thermo Fisher No. A18835, respectively.
    Thermo Fisher No.
    suggested: None
    Statistical analysis: All statistical analyses were carried out using Prism v9.1.0 (GraphPad, USA) and easyROC online calculator (http://www.biosoft.hacettepe.edu.tr/easyROC/).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    The performance of the tests was assessed by calculating sensitivity, specificity, and positive- and negative predictive values, associated P values and their 95% confidence intervals using Fisher exact test as implemented in GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Based on cutoff calculated in the ROC analysis for each test, samples were classified as positive or negative, and agreement between RT-PCR test and ELISAs was assessed using Cohen`s kappa statistic, calculated as described by Fleiss et al 200326, using an online calculator (GraphPad QuickCalcs, USA).
    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 presents however some limitations. The number of samples analyzed is still low. Additionally, to assess the relationship between positivity and evolution time we used the available date of RT-PCR confirmation, and this may not be a good surrogate of infection time. When available, studies should try to use the date of symptoms onset for better estimation of seroconversion time. It is also important to note that our COVID-19 cohort only include mild to moderate patients, and this fact may have influenced the strength of the immune response observed in our study. It has been shown that severity correlates with a more intense immune response and production of specific antibodies41,42,43,44,45. A more complete picture of the serology would have been obtained with patients from all the spectrum of the disease. Next steps should include testing of RT-PCR positive, asymptomatic subjects to characterize whether the tests are able to detect lower antibody levels likely expected in such situation. Nevertheless, our results confirm previous reports indicating the usefulness of these antigens for assays to measure seroconversion after SARS-CoV-2 infections22,46,47,48. The ELISA tests are simple to perform and very robust once optimized. Besides, since only a very small amount of sample is required, they are very convenient. Our results are consistent with those shown by others, suggesting that these serology assays have a very good potential for studies about measuring virus ex...

    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

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  3. Version published to 10.1101/2021.04.30.21256406 on medRxiv