From Multiplex Serology to Serolomics—A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

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1. Version published to 10.3390/v13050749

   Apr 24, 2021
2. 

   ScreenIT

   Oct 22, 2020

   SciScore for 10.1101/2020.10.19.20214916: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The study was approved by the Ethics Committee of the Medical Faculty Heidelberg (approval number S-148/2020) and conducted in accordance with the Declaration of Helsinki. Consent: Written informed consent was obtained from each participant.
   Randomization	We randomly selected n=88 participants to frequency match the age- and sex-distribution among the above-described hospitalized n=48 Covid-19 patients.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To assess potential cross-reactivity in the measured antibody responses, we included …

   SciScore for 10.1101/2020.10.19.20214916: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: The study was approved by the Ethics Committee of the Medical Faculty Heidelberg (approval number S-148/2020) and conducted in accordance with the Declaration of Helsinki. Consent: Written informed consent was obtained from each participant.
   Randomization	We randomly selected n=88 participants to frequency match the age- and sex-distribution among the above-described hospitalized n=48 Covid-19 patients.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   To assess potential cross-reactivity in the measured antibody responses, we included protein N of SARS-1 (NCBI accession no. NP_828858.1), and ccCoVs NL63 (NCBI accession no. YP_003771.1), 229E (NCBI accession no. NP_073556.1) , OC43 (NCBI accession no. AAT84358.1), and HKU1 (NCBI accession no. YP_173242.1) (Table 1).	HKU1 suggested: None
   Bound serum antibodies were labelled separately with biotinylated secondary antibodies (goat anti-human IgM/IgA/IgG, anti-human IgM, anti-human IgG, or anti-human IgA; Jackson ImmunoResearch, Westgrove, PA, USA) and subsequently incubated with Streptavidin-R-Phycoerythrin (MossBio, Pasadena, MD, USA).	anti-human IgM/IgA/IgG suggested: None anti-human IgM suggested: None anti-human IgG suggested: None anti-human IgA suggested: None
   Microscopy-based immunofluorescence detection of IgG antibodies to SARS-CoV-2: Microscopy-based immunofluorescence detection of IgG antibodies to SARS-CoV-2 antibodies was performed using a newly established semi-automated, semi quantitative approach, as described previously (Pape et al., 2020), for n=38 serum samples of hospitalized Covid-19 patients.	SARS-CoV-2 suggested: None
   Bound serum IgG antibodies to SARS-CoV-2 were detected using goat anti-human IgG-AlexaFluor 488 (Invitogen, Thermofisher Scientific) and evaluated by fluorescence microscopy.	anti-human IgG-AlexaFluor suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The technical minimum cut-off was 100 MFI, except for Protein S1 expressed in HEK293 cells (1000 MFI) due to observed variations in background values.	HEK293 suggested: None
   Briefly, fixed and permeabilized SARS-CoV-2 infected VeroE6 cells in 96-well plates were incubated with patient serum or control serum, respectively.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   The ESTHER study was approved by the ethics committees of the University of Heidelberg (approval number S-058/2000) and the medical board of the state of Saarland (approval number 67/00).	ESTHER suggested: (ESTHER, RRID:SCR_002621)
   All graphical and descriptive representations were performed using SAS 9.4 (SAS Institute, Cary, NC, USA) or GraphPad Prism 8 (Graphpad Software, Inc.	SAS Institute suggested: (Statistical Analysis System, RRID:SCR_008567) GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) Graphpad suggested: (GraphPad, RRID:SCR_000306)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Our study has several strengths and limitations. One major limitation is the fact that we only detected one out of 15 non-hospitalized Covid-19 patients as sero-positive. However, it should be noted that the non-hospitalized patients included in our study were all sampled within two weeks after symptom onset. In concordance with the literature (Okba et al., 2020), we observed that among hospitalized patients only after 14 days post symptom onset maximum sensitivity of the assay can be achieved. Moreover, non-hospitalized patients were of younger age and had a higher proportion of females than our control population, which might have affected the level of antibody response. A larger pre-pandemic control sample set with a wider age distribution is needed to address this question. The sensitivity among SARS-CoV-2 infected individuals experiencing mild or no symptoms also in relation to the time-point of infection needs to be further elucidated, especially with respect to future applications of the assay in large population-based cohorts. A major strength of our newly developed SARS-CoV-2 serological assay is its multiplex approach, allowing for the assessment of up to 100 antigens in parallel. It does not only permit inclusion of multiple SARS-CoV-2 proteins but also autoantigens or antigens from pathogens that might induce antibody responses due to (re-)activation or co-infection during the course of Covid-19 disease, as it was previously described for Herpesviruses (Amorim Dos...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

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   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.

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3. 

   Version published to 10.1101/2020.10.19.20214916 on medRxiv

   Oct 21, 2020