A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

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1. Version published to 10.1016/j.ebiom.2021.103348

   May 1, 2021
2. 

   ScreenIT

   Jan 21, 2021

   SciScore for 10.1101/2021.01.19.21249921: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: For ELISA validations, left-over sera from SARS-CoV-2 patients and sera from convalescent donors, as well as historical sera (<2020) were taken from the MedUni Wien Biobank, as approved by the ethics committee of the Medical University of Vienna (EK 1424/2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	The virus was originally isolated from a clinical specimen, a nasopharyngeal swab taken in mid-March 2020 from a 25-year old male patient in Lower Austria, and was further passaged twice on Vero E6 TMPRSS-2 cells in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (V/V)
   Cell Line Authentication	not …

   SciScore for 10.1101/2021.01.19.21249921: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: For ELISA validations, left-over sera from SARS-CoV-2 patients and sera from convalescent donors, as well as historical sera (<2020) were taken from the MedUni Wien Biobank, as approved by the ethics committee of the Medical University of Vienna (EK 1424/2020).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	The virus was originally isolated from a clinical specimen, a nasopharyngeal swab taken in mid-March 2020 from a 25-year old male patient in Lower Austria, and was further passaged twice on Vero E6 TMPRSS-2 cells in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (V/V)
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   5.1 Production of recombinant SARS-CoV-2 antigens for serodiagnosis: 5.2 Commercial antigen and antibody reagents: Recombinant spike proteins of the four common cold hCoV strains, HKU-1, OC43, NL63 and 229E were purchased from Sino Biological Inc, Beijing, CN (#40606-V08B, #40607-V08B, #40604-V08B and #40605-V08B, respectively).	HKU-1, suggested: None HKU-1, OC43 suggested: None NL63 suggested: None #40606-V08B suggested: None #40607-V08B suggested: None #40604-V08B suggested: None #40605-V08B suggested: None
   A recombinant chimeric human/mouse anti-SARS-CoV-2 NP antibody consisting of a mouse scFv fused to the Fc region of human IgG1 (clone 1A6) was purchased from Abcam, Cambridge, UK (#ab272852).	A recombinant chimeric human/mouse anti-SARS-CoV-2 NP antibody consisting suggested: None anti-SARS-CoV-2 NP suggested: None human IgG1 suggested: None
   Left-over samples were assessed for SARS-CoV-2 antibody levels and neutralizing titers in the early phase of infection and the study protocol was approved by the ethics committee of Upper Austria (EK1083/2020), in accordance with the Declaration of Helsinki.	SARS-CoV-2 suggested: None
   The calibrator set covered the concentration range 0 – 100 U/mL and concentrations of anti SARS-CoV-2 IgG antibodies recognizing either tRBD or NP in patient sera could be read directly from the calibration curve.	anti SARS-CoV-2 IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero E6 TMPRSS-2 cells, initially described in Hoffmann et al.(70) were kindly provided by Stefan Pöhlmann; Deutsches Primatenzentrum, Göttingen, Germany.	Vero E6 suggested: RRID:CVCL_XD71) TMPRSS-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   ROC-analysis data from automated tests (including Abbott ARCHITECT SARS-CoV-2 IgG, DiaSorin LIAISON®	Abbott suggested: (Abbott, RRID:SCR_010477)
   Data on the diagnostic performances of antigens and cross-reactivity were analyzed using Graphpad Prism Version 8.1.0	Graphpad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   (GraphPad Software, San Diego, CA, USA) Validation data were analyzed using MedCalc v19 (MedCalc Software, Ostend, Belgium) and Analyse-it 5.66 (Analyse-it Software, Leeds, UK) and SPSS 23.0 (SPSS Inc.).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798) SPSS suggested: (SPSS, RRID:SCR_002865)
   Data from SARS-CoV-2 acute sera from hospitalized individuals or outpatients obtained by the B&S Central Laboratory Linz were statistically analyzed with the MedCalc 13.1.2.0.	MedCalc suggested: (MedCalc, RRID:SCR_015044)

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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:

   A caveat of many assay validation studies is that performance characteristics are skewed by the exclusive inclusion of samples from hospitalized individuals, where robust antibody levels are to be expected (21). Likewise, the sole consideration of healthy donors in control groups may lead to overestimated assay specificity, as the impact of potential cross-reactive factors present in the general population is largely ignored. In this respect, auto-antibodies commonly found in individuals with inflammatory diseases (22) were already described to cross-react with SARS-CoV-1 antigens (23). To challenge our tests systems, we biased our large specificity cohort (n=1,126) by including samples with an increased propensity for cross-reactivity, including sera from individuals with inflammatory illnesses (n=359), sera from PCR-confirmed hCoV infections (n=8) and sera drawn during winter months to increase the likelihood of respiratory infections (n=494). Similarly, our sensitivity cohort (n=244) included convalescent sera from SARS-CoV-2-infected individuals covering the full spectrum of clinical manifestations (from asymptomatic to ICU patients). Among them 21% of the sera were collected from asymptomatic individuals or from individuals with mild to moderate illness, who may mount less robust and durable antibody responses after an infection (24). Based on these cohorts, we defined adequate test parameters to enable highly specific detection of SARS-CoV-2-specific antibodies. A cutof...

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04416100	Recruiting	Development of Interstitial Lung Disease (ILD) in Patients W…

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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 10.1101/2021.01.19.21249921v1 on medRxiv

   Jan 20, 2021