Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

  1. Livia Mazzini
  2. Donata Martinuzzi
  3. Inesa Hyseni
  4. Giulia Lapini
  5. Linda Benincasa
  6. Pietro Piu
  7. Claudia Maria Trombetta
  8. Serena Marchi
  9. Ilaria Razzano
  10. Alessandro Manenti
  11. Emanuele Montomoli

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Abstract

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 18 million cases of disease and 700,000 deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays aimed at detecting different classes of antibodies constitute the best surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population, in order to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to know the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict population immunity and possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, we performed a subtyping Immunoglobulin G ELISA. Our data showed an excellent statistical correlation between the neutralization titer and the IgG, IgM and IgA ELISA response against the receptor-binding domain of the spike protein, confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories which do not have Biosecurity level-3 facilities.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.10.243717: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Human IgG1 anti-SARS-CoV-2 Spike (S1) Antibody CR3022 (Native Antigen, place)
    S1
    suggested: (Abcam Cat# ab273074, RRID:AB_2847846)
    , Human IgM anti-SARS-CoV-2 Spike (S1) Antibody CR3022 (Native Antigen, Oxford, UK) and anti-Spike RBD (SARS-CoV-2/COVID 19) human monoclonal antibody (eEnzyme, Gaithersburg, USA) were used as positive controls in ELISA.
    Human IgM anti-SARS-CoV-2 Spike ( S1
    suggested: None
    Antibody CR3022 ( Native Antigen , Oxford , UK )
    suggested: None
    anti-Spike RBD
    suggested: (MyBioSource Cat# MBS434247, RRID:AB_2848131)
    Three human serum samples containing heterologous neutralizing antibodies, provided by NIBSC, were used to verify the specificity of the ELISA: WHO 1st International Standard for Pertussis antiserum (lot. 06/140); WHO 2nd International Standard for antibody to influenza H1N1pdm virus (lot. 10/202); WHO 1st International Standard for Diphtheria Antitoxin (lot: 10/262).
    Antitoxin
    suggested: None
    SARS-CoV-2 purified antigen, live virus and titration: Five different purified recombinant S proteins (S1 and RBD domain) were tested for their ability to detect specific human antibodies: S1-SARS-CoV-2 (HEK293) Cod.
    S1-SARS-CoV-2 ( HEK293
    suggested: None
    ELISA): Specific anti-SARS-CoV-2 IgG antibodies were detected by means of two commercial ELISA kits: Euroimmun and Eagle Biosciences.
    anti-SARS-CoV-2 IgG
    suggested: None
    Next, after the washing step, 100 µl/well of Goat anti-Human IgG-Fc HRP-conjugated antibody or IgM (µ-chain) and IgA (α-chain) diluted 1:100,000 or 1:100,000 and 1:75,000, respectively, (Bethyl Laboratories, Montgomery USA) were added.
    anti-Human IgG-Fc
    suggested: None
    IgM
    suggested: None
    α-chain
    suggested: (Sigma-Aldrich Cat# M8695, RRID:AB_2617179)
    In-House RBD Enzyme-Linked Immunosorbent Assay (ELISA) IgG1, IgG2, IgG3 and IgG4: An indirect ELISA was performed in order to determine the RBD-specific IgG1, IgG2, IgG3 and IgG4 antibody concentration in serum samples [38].
    In-House RBD Enzyme-Linked Immunosorbent Assay ( ELISA ) IgG1
    suggested: None
    IgG1
    suggested: None
    IgG3
    suggested: None
    IgG4: An indirect ELISA was performed in order to determine the RBD-specific IgG1 , IgG2 , IgG3 and IgG4 antibody concentration in serum samples [ 38]
    suggested: None
    the RBD-specific IgG1
    suggested: None
    RBD-specific IgG1
    suggested: None
    IgG2
    suggested: None
    IgG4 antibody concentration in serum samples [ 38]
    suggested: None
    IgG4
    suggested: None
    Mouse anti-human IgG1, IgG2, IgG3 and IgG4 Fc-HRP (Southern Biotech, USA) secondary antibodies were used at 1:8000 dilution.
    anti-human
    suggested: None
    anti-human IgG1
    suggested: (Innovative Research Cat# MHCD8000, RRID:AB_1485866)
    IgG2, IgG3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    VERO E6 cells were seeded in a 96-well plate using D-MEM high glucose 2% FBS at a density of 1.5 x 106cells per well, in order to obtain a 70-80% sub-confluent cell monolayer after 24 hours.
    VERO E6
    suggested: None
    SCV2-S1-150P (eEnzyme Gaithersburg, MD, USA); S1-SARS-CoV-2 (HEK293) Cod.
    S1-SARS-CoV-2
    suggested: None
    In-House S1 and RBD Enzyme-Linked Immunosorbent Assay (ELISA) IgG, IgM and IgA: ELISA plates were coated with 1µg/mL of purified recombinant Spike S1 Protein (aa 18-676) (eEnzyme, Gaithersburg, MD, USA) or with 1µg/mL Spike-RBD (Arg319-Phe541) (Sino Biological, China), both expressed and purified from HEK 293 cells.
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    ELISA): Specific anti-SARS-CoV-2 IgG antibodies were detected by means of two commercial ELISA kits: Euroimmun and Eagle Biosciences.
    Eagle Biosciences
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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. ScreenIT

    SciScore for 10.1101/2020.08.10.243717: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Each protein was evaluated on using three coating concentrations (1, 2 and 3 µg/mL) and four different dilutions of the secondary Horse Radish Peroxidase (HRP) conjugate anti-human IgG, IgM and IgA antibodies.
    anti-human IgG
    suggested: None
    As a test control, human IgG1 monoclonal antibody (mAb) anti-SARS-CoV-2 spike (S1) (CR3022 Native antigen), human IgM mAb anti-SARS-CoV-2 spike (S1) (CR3022 Absolute antibody) and human IgG1 anti-Spike RBD (SCV2-RBD eEnzyme) were used.
    anti-SARS-CoV-2
    suggested: (Abcam Cat# ab273074, RRID:AB_2847846)
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    human IgG1
    suggested: None
    Human IgG1 anti-SARS-CoV-2 Spike (S1) Antibody CR3022 (Native Antigen, place)
    S1
    suggested: (Abcam Cat# ab273074, RRID:AB_2847846)
    , Human IgM anti-SARS-CoV-2 Spike (S1) Antibody CR3022 (Native Antigen, Oxford, UK) and anti-Spike RBD (SARS-CoV-2/COVID 19) human monoclonal antibody (eEnzyme, Gaithersburg, USA) were used as positive controls in ELISA.
    Human IgM anti-SARS-CoV-2 Spike ( S1
    suggested: None
    Antibody CR3022 ( Native Antigen , Oxford , UK )
    suggested: None
    anti-Spike RBD
    suggested: (MyBioSource Cat# MBS434247, RRID:AB_2848131)
    Three human serum samples containing heterologous neutralizing antibodies, provided by NIBSC, were used to verify the specificity of the ELISA: WHO 1st International Standard for Pertussis antiserum (lot. 06/140); WHO 2nd International Standard for antibody to influenza H1N1pdm virus (lot. 10/202); WHO 1st International Standard for Diphtheria Antitoxin (lot: 10/262).
    Antitoxin
    suggested: None
    ELISA): Specific anti-SARS-CoV-2 IgG antibodies were detected by means of two commercial ELISA kits: Euroimmun and Eagle Biosciences.
    anti-SARS-CoV-2 IgG
    suggested: None
    Next, after the washing step, 100 µl/well of Goat anti-Human IgG-Fc HRP-conjugated antibody or IgM (µ-chain) and IgA (α- chain) diluted 1:100,000 or 1:100,000 and 1:75,000, respectively, (Bethyl Laboratories, Montgomery USA) were added.
    anti-Human IgG-Fc
    suggested: None
    IgM
    suggested: None
    IgA
    suggested: None
    In-House RBD Enzyme-Linked Immunosorbent Assay (ELISA) IgG1, IgG2, IgG3 and IgG4: An indirect ELISA was performed in order to determine the RBD-specific IgG1, IgG2, IgG3 and IgG4 antibody concentration in serum samples [38].
    In-House RBD Enzyme-Linked Immunosorbent Assay ( ELISA ) IgG1
    suggested: None
    IgG1
    suggested: None
    IgG4: An indirect ELISA was performed in order to determine the RBD-specific IgG1 , IgG2 , IgG3 and IgG4 antibody concentration in serum samples [ 38]
    suggested: None
    the RBD-specific IgG1
    suggested: None
    RBD-specific IgG1
    suggested: None
    IgG2
    suggested: None
    IgG3
    suggested: None
    IgG4 antibody concentration in serum samples [ 38]
    suggested: None
    Mouse anti-human IgG1, IgG2, IgG3 and IgG4 Fc-HRP (Southern Biotech, USA) secondary antibodies were used at 1:8000 dilution.
    anti-human
    suggested: None
    IgG2, IgG3
    suggested: None
    IgG4
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    VERO E6 cells were seeded in a 96-well plate using D-MEM high glucose 2% FBS at a density of 1.5 x 106cells per well, in order to obtain a 70-80% sub-confluent cell monolayer after 24 hours.
    VERO E6
    suggested: None
    In-House S1 and RBD Enzyme-Linked Immunosorbent Assay (ELISA) IgG, IgM and IgA: ELISA plates were coated with 1µg/mL of purified recombinant Spike S1 Protein (aa 18-676) (eEnzyme, Gaithersburg, MD, USA) or with 1µg/mL Spike-RBD (Arg319-Phe541) (Sino Biological, China), both expressed and purified from HEK 293 cells.
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    ELISA): Specific anti-SARS-CoV-2 IgG antibodies were detected by means of two commercial ELISA kits: Euroimmun and Eagle Biosciences.
    Eagle Biosciences
    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 Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    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 10.1101/2020.08.10.243717 on bioRxiv