A scalable serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination

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1. 

   Version published to 10.1101/2021.10.25.21265476v2 on medRxiv

   Feb 26, 2022
2. Version published to 10.1002/cti2.1380

   Jan 1, 2022
3. 

   ScreenIT

   Oct 29, 2021

   SciScore for 10.1101/2021.10.25.21265476: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Participant recruitment and study approval – Toronto cohorts: Negative control serum samples were from patients enrolled in cancer studies pre-COVID-19 (prior to November 2019; Mount Sinai Hospital (MSH) Research Ethics Board (REB) studies #01-0138-U and #01-0347-U), which were archived and frozen in the Lunenfeld-Tanenbaum Research Institute (LTRI
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Recombinant antibody production: The llama single domain antibody (VHH) VHH72-hFc1X7 (VHH72-Fc) was described previously (PDB entry 6WAQ_1) (17); …

   SciScore for 10.1101/2021.10.25.21265476: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Participant recruitment and study approval – Toronto cohorts: Negative control serum samples were from patients enrolled in cancer studies pre-COVID-19 (prior to November 2019; Mount Sinai Hospital (MSH) Research Ethics Board (REB) studies #01-0138-U and #01-0347-U), which were archived and frozen in the Lunenfeld-Tanenbaum Research Institute (LTRI
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Recombinant antibody production: The llama single domain antibody (VHH) VHH72-hFc1X7 (VHH72-Fc) was described previously (PDB entry 6WAQ_1) (17); additional VHHs (NRCoV2-04 and NRCoV2-20) were isolated in-house from llamas immunized with recombinant SARS-CoV-2 trimeric spike ectodomain SmT1 (Supplementary Figure 2).	NRCoV2-20 suggested: None
   VHH sequences were fused to an antibody-dependent cell-mediated cytotoxicity (ADCC)-attenuated human IgG1 Fc domain (hFc1X7, from patent US 2019 352 383A1).	human IgG1 Fc domain ( hFc1X7 suggested: None
   The anti-human-IgG monoclonal antibodies (mAbs) IgG#5 and IgG#6 were derived from mice immunized with human IgG; heavy chain (HC) and light chain (LC) variable domain sequences (VH and VL) were fused to mouse IgG2a and mouse kappa LC constant sequences, respectively, to express full-length mAbs.	anti-human-IgG suggested: None mouse IgG2a suggested: None
   SARS-CoV-2 antibody-negative blood was spotted directly from EDTA Vacutainer tubes onto DBS cards.	SARS-CoV-2 suggested: None
   All matched plasma and contrived DBS samples were tested using the anti-SARS-CoV-2 ELISA IgG kit (EUROIMMUN, Lübeck, Germany), according to the manufacturer’s instructions, to verify that donors were either positive or negative for SARS-CoV-2 antibodies prior to shipping to Toronto and Ottawa.	anti-SARS-CoV-2 ELISA IgG suggested: None
   For spike and its RBD, the recombinant antibodies used were VHH72-Fc IgG (NRC; see above), human anti-spike S1 IgG (clone HC2001, GenScript, #A02038), human anti-Spike S1 IgM (clone hIgM2001, GenScript, #A02046), and human anti-spike IgA (clone CR3022, Absolute Antibody, Oxford, United Kingdom, #Ab01680-16.0).	anti-spike S1 IgG suggested: None anti-Spike S1 IgM suggested: None anti-spike IgA suggested: None
   For N, the antibodies used were human anti-nucleocapsid IgG (clone HC2003, GenScript, #A02039)	anti-nucleocapsid IgG suggested: None
   anti-nucleoprotein IgM (CR3018 (03-018), Absolute Antibody, #Ab01690 -15.0), and anti-nucleoprotein IgA (CR3018 (03-018), Absolute Antibody, #Ab01690 -16.0).	anti-nucleoprotein IgM suggested: None anti-nucleoprotein IgA suggested: None CR3018 suggested: (Imported from the IEDB Cat# CR3018, RRID:AB_2833185)
   Negative control antibodies purified from human serum (final 1 µg/mL; human IgG, Sigma-Aldrich, Oakville, ON, Canada,	human IgG suggested: None
   Anti-human secondary antibodies (recombinant anti-human IgG#5-HRP, goat anti-human IgG Fcy-HRP (Jackson ImmunoResearch Labs, West Grove, PA, USA, #109-035-098)	Anti-human secondary suggested: None anti-human IgG#5-HRP suggested: (Jackson ImmunoResearch Labs Cat# 109-035-098, RRID:AB_2337586) anti-human IgG Fcy-HRP suggested: None
   Briefly, 100 μL samples of titrations of anti-SARS-CoV-2 S CR3022 Human IgG1 (Absolute Antibody, Ab01680-10.0), anti-SARS-CoV-2 S CR3022 Human IgA (Absolute Antibody, Ab01680-16.0), or anti-SARS-CoV-2 S CR3022 Human IgM (Absolute Antibody, Ab01680-15.0) were diluted in 1% w/v skim milk in PBST.	anti-SARS-CoV-2 S CR3022 Human IgG1 suggested: None Human suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080) anti-SARS-CoV-2 S CR3022 Human IgA suggested: None anti-SARS-CoV-2 S CR3022 Human IgM suggested: None
   The final isotype-specific secondary antibodies used were anti-human IgG#5-HRP (Supplementary Figure 3), anti-human IgA-HRP (Jackson ImmunoResearch Labs, 109-035-011), and anti-human IgM-HRP (Jackson ImmunoResearch Labs, 109-035-129).	anti-human IgA-HRP suggested: (SouthernBiotech Cat# 2050-05, RRID:AB_2687526)
   Two concentrations of secondary antibody IgG#5-HRP (0.09 and 0.18 μg/mL) were assessed using dilution curves of the VHH72-Fc antibody (to detect spike and its RBD) or an anti-N antibody (to detect N; Supplementary Figure 7), and the best concentration (0.18 μg/mL) was further tested on a dilution series of 32 serum samples provided by CBS (Supplemental Figure 8).	VHH72-Fc suggested: None anti-N suggested: None
   We also tested anti-RBD NRCoV2-04 and NRCoV2-20 recombinant calibration antibodies, which were comparable to VHH72-Fc in reference curves (Supplementary Figure 7).	anti-RBD suggested: None NRCoV2-04 suggested: None
   For chemiluminescent assays, 10 µL of goat anti-human IgM-HRP (1:10,000; 0.80 ng/well) or goat anti-human IgA-HRP (1:12,000, 0.66 ng/well) were used as secondary antibodies.	anti-human IgM-HRP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Protein production: Spike trimer: The SARS-CoV-2 spike ectodomain construct (SmT1) with S1/S2 furin site mutations, K986P/V987P prefusion-stabilizing mutations, and human resistin as a trimerization partner (15) was produced using stably transfected Chinese Hamster Ovary (CHO) pools (CHOBRI/2353™ cells) and purified as described (8).	CHOBRI/2353™ suggested: None
   The construct was expressed by transient gene expression in CHOBRI/55E1™ cells as described above (15).	CHOBRI/55E1™ suggested: None
   VHH and mAb sequences were synthesized by GenScript using C. griseus codon bias for expression in CHO cells and cloned into the pTT5™ plasmid.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   The ACE2-BAP cDNA was expressed by transient gene expression in CHOBRI/55E1 cells as described (15) with the addition of 5% (w/w) pTT5™-BirA (an Escherichia coli biotin ligase) expression plasmid as described previously (18).	CHOBRI/55E1 suggested: None
   Recombinant DNA
   Sentences	Resources
   Nucleocapsid: N cDNA (corresponding to amino acids 1–419 of YP_009724397) was synthesized by GenScript (Piscataway, NJ, USA; using Cricetulus griseus codon bias) with a C-terminal FLAG-Twin-Strep-tag-(His)6 tag and cloned into the pTT5 expression plasmid (NRC) to create NCAP (16).	pTT5 suggested: RRID:Addgene_52326)
   Amino acids 331–521 of the SARS-CoV-2 spike protein (YP_009724390.1) corresponding to the RBD were cloned into the pTT5™ vector using EcoRI and BamHI.	pTT5™ suggested: None
   The ACE2-BAP cDNA was expressed by transient gene expression in CHOBRI/55E1 cells as described (15) with the addition of 5% (w/w) pTT5™-BirA (an Escherichia coli biotin ligase) expression plasmid as described previously (18).	pTT5™-BirA suggested: None
   Software and Algorithms
   Sentences	Resources
   Ottawa: Samples from DBS cards were punched manually or in a semi-automated manner using a PerkinElmer DBS puncher (PerkinElmer, Woodbridge, ON, Canada; 3.2 mm discs) or a BSD600 Ascent puncher (BSD Robotics; 3 mm discs) and eluted in 100 μL/disc PBS + 1% Triton X-100 for up to 16 h (minimum 4 h) in 96-well U-bottom plates on a shaker at room temperature.	Canada suggested: (Brain Canada, RRID:SCR_005053)
   Other data analyses: Plots were generated in R using the ggplot2, lattice, latticeExtra, grid, and gridExtra packages.	ggplot2 suggested: (ggplot2, RRID:SCR_014601)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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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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4. 

   Version published to 10.1101/2021.10.25.21265476v1 on medRxiv

   Oct 26, 2021