Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients
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Abstract
Saliva is an alternative biofluid to serum for detecting and monitoring IgG to SARS-CoV-2 spike and RBD antigens in COVID-19.
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SciScore for 10.1101/2020.08.01.20166553: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 4–12 weeks PSO.
IRB: All samples were collected after Research Ethics Board (REB) review (see Sample section above for the individual REB approval numbers).Randomization No randomization was performed, since this is an observational study. 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 This observational study focused on monitoring the levels of … SciScore for 10.1101/2020.08.01.20166553: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 4–12 weeks PSO.
IRB: All samples were collected after Research Ethics Board (REB) review (see Sample section above for the individual REB approval numbers).Randomization No randomization was performed, since this is an observational study. 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 This observational study focused on monitoring the levels of antibodies to SARS-CoV-2 antigens in serum and saliva of patients with confirmed SARS-CoV-2 infection. SARS-CoV-2suggested: NoneAfter washing 4 times, 10 μl of one of the following secondary antibodies (all from Jackson ImmunoResearch) diluted in 1% BLOTTO in PBS-T were added at the indicated concentrations followed by incubation for 2 hr at room temperature: Goat anti-human IgG Fcy – HRP (#109-035-098; 1:40,000 or 0.2 ng per well), Goat anti-human IgM Fc5u – HRP (#109035-129; 1:12,000 or 0.66 ng per well) or Goat anti-human IgA a chain – HRP (#109-035-127; 1:10,000 or 0.8 ng per well). anti-human IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-035-098, RRID:AB_2337586)anti-human IgMsuggested: (Jackson ImmunoResearch Labs Cat# 109-035-129, RRID:AB_2337588)anti-human IgAsuggested: NoneAntibodies used for the standard curves were: Human anti-spike S1 IgG (A02038, GenScript), anti-spike S1 IgM (A02046, GenScript) and Ab01680 anti-spike IgA (Ab01680-16, Absolute Antibody), all used at 0.5 to 10ng per well. anti-spike S1 IgGsuggested: NoneA02038suggested: Noneanti-spike S1 IgMsuggested: NoneA02046suggested: (GenWay Biotech Inc. Cat# GWB-A02046, RRID:AB_10276779)anti-spike IgAsuggested: NoneAnti-human Ig antibody (Southern Biotech, 2010–01) diluted 1:1000 in PBS was added to 96-well Nunc MaxiSorp™ plates (ThermoFisher, 44-2404-21). Anti-human Ig antibodysuggested: NoneAnti-human Igsuggested: NoneHRP-conjugated secondary antibodies against IgA, IgG, and IgM (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053–05, IgG: 2044–05, IgM: 2023–05) were added to the appropriate wells at 1:1000 in 2.5% BLOTTO and incubated for 1 hour at 37°C. HRP-conjugated secondary antibodies against IgA, IgG, and IgM (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053–05, IgG: 2044–05, IgM: 2023–05)suggested: NoneHRP-conjugated secondary antibodies against IgA, IgGsuggested: Nonegoat anti-human IgA-suggested: (InvivoGen Cat# fab-iga, RRID:AB_11125122)Horseradish peroxidase (HRP)-conjugated Goat anti human-IgG, IgA, and anti-IgM secondary antibodies (Southern Biotech, IgG: 2044–05, IgA: 2053–05, IgM: 2023–05) were added to wells at dilutions of 1:1000, 1:2000 and 1:1000 in 2.5% BLOTTO, respectively, and incubated for 1 hour at 37°C. anti human-IgG, IgA,suggested: Noneanti human-IgG, IgAsuggested: Noneanti-IgMsuggested: (SouthernBiotech Cat# 2023-01, RRID:AB_2795619)For serum samples, seven multivariable linear regression models were constructed (one for each of anti-RBD IgA, anti-S IgA, anti-RBD IgG, anti-S IgG, anti-RBD IgM, anti-spike IgM, neutralizing antibody). anti-RBD IgAsuggested: Noneanti-S IgAsuggested: Noneanti-RBD IgGsuggested: Noneanti-S IgGsuggested: Noneanti-RBD IgMsuggested: Noneanti-spike IgMsuggested: NoneDevelopment and validation of manual colorimetric and automated chemiluminescent assays for monitoring RBD and spike trimers antibodies in serum or plasma. Fig. S2. S2suggested: NoneEffect of heat versus detergent inactivation of saliva samples on the detection of anti-RBD antibodies in a manual, colourimetric ELISA. Fig. S5. anti-RBDsuggested: NoneS5suggested: NoneExperimental Models: Cell Lines Sentences Resources To stabilize the spike protein in a trimeric form, the cDNA was cloned in-frame with the human resistin cDNA (aa 23–108) containing a C-terminal FLAG-(His)6 tag (Cricetulus griseus codon bias, GenScript) into a modified cumate-inducible pTT241 expression plasmid and transfected in CHO2353 cells (Stuible et al, manuscript in preparation) followed by methionine sulfoximine selection for 14 days to generate a stable CHO pool. CHO2353suggested: NoneViral stock was expanded using Vero E6 as previously described such that stored aliquots of stock contain 2% FBS. Vero E6suggested: RRID:CVCL_XD71)Initial experiments were done with Triton X-100 (Sigma-Aldrich) serially diluted and applied to Vero-E6 cells in 96-well flat bottom plates to determine the minimum concentration required to prevent toxicity to cells. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Spike trimer was expressed as follows: the SARS-CoV-2 spike sequence (aa 1–1208 from Genebank accession number MN908947 with the S1/S2 furin site (residues 682–685) mutated [RRAR->GGAS] and K986P / V987P stabilizing mutations was codon-optimized (Cricetulus griseus codon bias) and synthesized by Genscript. Genebanksuggested: NoneThese analyses were performed in Prism (GraphPad), Version 8.3. Prismsuggested: (PRISM, RRID:SCR_005375)References and Notes: Notessuggested: NoneResults 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 did not find any issues relating to the usage of bar graphs.
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.
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SciScore for 10.1101/2020.08.01.20166553: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 412 weeks post onset of symptoms. Randomization not detected. 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 After washing 4 times, 10 µl of one of the following secondary antibodies (all from Jackson ImmunoResearch) diluted in 1% BLOTTO in PBS-T were added at the indicated concentrations followed by incubation for 2 hr at room temperature: Goat anti-human IgG Fcy … SciScore for 10.1101/2020.08.01.20166553: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 412 weeks post onset of symptoms. Randomization not detected. 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 After washing 4 times, 10 µl of one of the following secondary antibodies (all from Jackson ImmunoResearch) diluted in 1% BLOTTO in PBS-T were added at the indicated concentrations followed by incubation for 2 hr at room temperature: Goat anti-human IgG Fcy – 035-129; 1:12,000 or 0.66 ng per well) or Goat anti-human IgA a chain - HRP (#109-035-127; 1:10,000 or 0.8 ng per well). anti-human IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-035-127, RRID:AB_2337587)anti-human IgAsuggested: NoneAntibodies used for the standard curves were: Human anti-spike S1 IgG (A02038, GenScript), anti-spike S1 IgM (A02046, GenScript) and Ab01680 anti-spike IgA (Ab01680-16, Absolute Antibody), all used at 0.5 to 10ng per well. anti-spike S1 IgGsuggested: NoneA02038suggested: Noneanti-spike S1 IgMsuggested: NoneA02046suggested: (GenWay Biotech Inc. Cat# GWB-A02046, RRID:AB_10276779)anti-spike IgAsuggested: NoneAnti-human Ig antibody (Southern Biotech, 2010-01) diluted 1:1000 in PBS was added to 96-well Nunc MaxiSorp™ plates (ThermoFisher, 44-2404-21). Anti-human Ig antibodysuggested: (SouthernBiotech Cat# 2010-01, RRID:AB_2687525)Anti-human Igsuggested: (SouthernBiotech Cat# 2010-01, RRID:AB_2687525)HRP-conjugated secondary antibodies against IgA and IgG (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053-05, IgG: 2044-05) were added to the appropriate wells at 1:1000 in 2.5% BLOTTO and incubated for 1 hour at 37oC. HRP-conjugated secondary antibodies against IgA and IgG (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053-05, IgG: 2044-05)suggested: NoneHRP-conjugated secondary antibodies against IgA and IgGsuggested: Nonegoat anti-human IgA-suggested: (InvivoGen Cat# hrp-iga, RRID:AB_11124937)Horseradish peroxidase (HRP)-conjugated Goat anti human-IgA and anti-IgG secondary antibodies (Southern Biotech, 2053-05 and 2044-05) were added to wells at dilutions of 1:2000 and 1:1000 in 2.5% BLOTTO, respectively, and incubated for 1 hour at 37oC. Goat anti human-IgAsuggested: Noneanti human-IgAsuggested: Noneanti-IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources To stabilize the spike protein in a trimeric form, the cDNA was cloned in-frame with the human resistin cDNA (aa 23-108) containing a Cterminal FLAG-(His)6 tag (Cricetulus griseus codon bias, GenScript) into a modified cumateinducible pTT241 expression plasmid and transfected in CHO2353 cells (Stuible et al., CHO2353suggested: NoneViral stock was expanded using Vero E6 as previously described such that stored aliquots of stock contain 2% FBS. Vero E6suggested: RRID:CVCL_XD71)Initial experiments were done with Triton X-100 (Sigma-Aldrich) serially diluted and applied to Vero-E6 cells in 96-well flat bottom plates to determine the minimum concentration required to prevent toxicity to cells. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Antigen production – serum assays Spike trimer was expressed as follows: the SARS-CoV-2 spike sequence (aa 1-1208 from Genebank accession number MN908947 with the S1/S2 furin site (residues 682–685) mutated [RRAR->GGAS] and K986P / V987P stabilizing mutations was codon-optimized (Cricetulus griseus codon bias) and synthesized by Genscript. Genebanksuggested: NoneA fourparameter logistic curve was used to determine the line of best fit for the standard curve, and sample Ig quantities were interpolated accordingly, using Prism Graphpad, Version 8.3. Graphpadsuggested: (GraphPad, RRID:SCR_000306)All analysis was performed in SAS 9.4M6 (SAS Institute, Cary, NC). SAS Institutesuggested: (Statistical Analysis System, RRID:SCR_008567)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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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