Development and evaluation of low-volume tests to detect and characterise antibodies to SARS-CoV-2

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1. 

   Version published to 10.1101/2022.05.03.22274395v2 on medRxiv

   May 16, 2022
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   ScreenIT

   May 8, 2022

   SciScore for 10.1101/2022.05.03.22274395: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: PCR confirmed and clinically suspected severe COVID-19 cases admitted to hospital were recruited into the DISCOVER study at North Bristol NHS Trust for which HRA Approval was granted by the South Yorkshire Research Ethics Committee (20/YH/0121). Consent: All samples were used in accordance with the Human Tissue Act (2004) with appropriate consent and ethical approvals in place.
   Sex as a biological variable	not detected.
   Randomization	Blinding of validation set: The validation set of samples (n=807) were split into multiple aliquots (n=5) for randomisation and blinding by assigning a new barcode ID for each aliquot.
   Blinding	Blinding of validation set: The validation set of samples (n=807) …

   SciScore for 10.1101/2022.05.03.22274395: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: PCR confirmed and clinically suspected severe COVID-19 cases admitted to hospital were recruited into the DISCOVER study at North Bristol NHS Trust for which HRA Approval was granted by the South Yorkshire Research Ethics Committee (20/YH/0121). Consent: All samples were used in accordance with the Human Tissue Act (2004) with appropriate consent and ethical approvals in place.
   Sex as a biological variable	not detected.
   Randomization	Blinding of validation set: The validation set of samples (n=807) were split into multiple aliquots (n=5) for randomisation and blinding by assigning a new barcode ID for each aliquot.
   Blinding	Blinding of validation set: The validation set of samples (n=807) were split into multiple aliquots (n=5) for randomisation and blinding by assigning a new barcode ID for each aliquot.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Purified proteins were analysed by SDS-PAGE and by Western-blots assays using an anti-His tag antibody (Sigma).	anti-His tag suggested: None
   After washing, HRP-conjugated anti-human Pan-Immunoglobulin (Pan) (Sigma), IgG (Southern Biotech), IgA (Sigma) or IgM (Sigma) secondary antibody, in the same dilution buffer as the samples, was added (50 µl per well) and incubated for 1 hour.	IgG ( Southern Biotech) suggested: (SouthernBiotech Cat# 1050-01, RRID:AB_2737431) IgA ( Sigma ) suggested: (Sigma-Aldrich Cat# I1010, RRID:AB_1163625) IgM ( Sigma ) secondary antibody suggested: None IgM suggested: None
   Roche SARS-CoV-2 anti-nucleocapsid antibody assay: Serum samples from PCR-confirmed cases were analysed using the commercial Elecsys® Anti-SARS-CoV-2 (Roche) in the Department of Microbiology, Infection Sciences, Southmead Hospital, North Bristol NHS Trust, Southmead Road, BS10 5NB, UK following manufacturer’s instructions.	anti-nucleocapsid suggested: None Anti-SARS-CoV-2 suggested: None
   Transfected 293T cells were then infected with VSV*G-FLuc particles for 2 hours, washed with PBS, then incubated with fresh DMEM, supplemented with 10% FBS and 1:2000 (v/v) I1 (anti-VSV-G) antibody (absolute antibody Ab01401-10.3).	I1 suggested: None anti-VSV-G suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   VSV-G-harbouring BHK21 cells were infected with VSV*ΔG- FLuc particles to generate complemented VSV*G-FLuc particles as previously described (35).	BHK21 suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)
   , 293T cells were seeded and transiently transfected with a plasmid corresponding to the original Wuhan strain Spike protein (pCAGGS-S2-spike) using Turbofect transfection reagent (ThermoFisher R0532) for 16 hours following the manufacturer’s instructions.	293T suggested: None
   Optimal pseudotype cell entry was achieved using VeroE6 cells stably expressing the human angiotensin-converting enzyme 2 (ACE2) receptor and the cell surface protease TMPRSS2 (Vero ACE2 TMPRSS2 (VAT) cells, which were a kind gift from Dr Suzannah Rihn, MRC-University of Glasgow Centre for Virus Research (36)	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49) Vero ACE2 suggested: RRID:CVCL_A7UJ)
   Recombinant DNA
   Sentences	Resources
   The sequence was synthesized with an NdeI restriction site at the 5’ end and the BamHI site at the 3’ end and cloned into pET28a expression vector.	pET28a suggested: RRID:Addgene_139598)
   The recombinant plasmids (pET28a-NP-FL) were transformed into E. coli strain BL21 (DE3)	pET28a-NP-FL suggested: None
   , 293T cells were seeded and transiently transfected with a plasmid corresponding to the original Wuhan strain Spike protein (pCAGGS-S2-spike) using Turbofect transfection reagent (ThermoFisher R0532) for 16 hours following the manufacturer’s instructions.	pCAGGS-S2-spike suggested: None
   Software and Algorithms
   Sentences	Resources
   Images were acquired on the ImageXpress Pico Automated Cell Imaging System (Molecular Devices) using a 10X objective and infected cells detected and quantified using Cell ReporterXpress software (Molecular Devices).	Cell ReporterXpress suggested: None
   Statistical Analysis: Data analyses were performed using either R software with R Studio, and GraphPad Prism (version 9) as detailed below.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   However, sample readouts using other methods including interpolated unit values (from a 4- parameter logistic regression model fit (on Prism or within BMG software) to the 7- point standard pool dilution series) and AUC from sample dilution series were used in the development stage.	Prism suggested: (PRISM, RRID:SCR_005375)

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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:

   Strengths of this study include rigorous development of high performance, low blood volume, cost-effective tests which can be easily deployed in a variety of settings, but our approach also has several limitations. Firstly, whilst samples from pre-pandemic children were included, samples from children with COVID-19 were not available to us and as such, assay performance for detecting recent paediatric infections cannot be reported. However, since widespread vaccination of children is not currently common in many countries while asymptomatic/mild paediatric infections are, antibody assays offer a useful tool for monitoring infection in this age group. The antigens used in the in-house assays were generated using the genetic sequence from the parent Wuhan strain of SARS-CoV-2 first described in 2020 (7) from which several new variants of concern (VOC) have evolved and have caused significant waves of infection globally. Some of these variants, especially Omicron, include multiple mutations in these target antigens and as such, may lead to antibody responses with differential binding to the target antigens. Indeed, antibodies responses raised to antigens from one SARS-CoV-2 variant genetic sequence lead to differential ability to neutralise VOC strains. However, whilst others have shown reduced binding to antigens from sequences of VOCs, rates of seropositivity when using different antigens, and/or from people who were infected with non-Wuhan variants, appear to be relatively un...

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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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.05.03.22274395v1 on medRxiv

   May 5, 2022