Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples
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SciScore for 10.1101/2021.07.02.450915: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: These samples were drawn from Seattle Children’s workforce and adult community members diagnosed with COVID-19 by detection of SARS-CoV-2 nucleic acids in nasopharyngeal specimens, who subsequently enrolled into the Seattle Children’s SARS2 Recovered Cohort with informed consent and Institutional Review Board approval through SCRI.
IRB: These samples were drawn from Seattle Children’s workforce and adult community members diagnosed with COVID-19 by detection of SARS-CoV-2 nucleic acids in nasopharyngeal specimens, who subsequently enrolled into the Seattle Children’s SARS2 Recovered Cohort with informed consent and Institutional Review Board approval through SCRI.Sex as a biological … SciScore for 10.1101/2021.07.02.450915: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: These samples were drawn from Seattle Children’s workforce and adult community members diagnosed with COVID-19 by detection of SARS-CoV-2 nucleic acids in nasopharyngeal specimens, who subsequently enrolled into the Seattle Children’s SARS2 Recovered Cohort with informed consent and Institutional Review Board approval through SCRI.
IRB: These samples were drawn from Seattle Children’s workforce and adult community members diagnosed with COVID-19 by detection of SARS-CoV-2 nucleic acids in nasopharyngeal specimens, who subsequently enrolled into the Seattle Children’s SARS2 Recovered Cohort with informed consent and Institutional Review Board approval through SCRI.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 HRP conjugated rec-Protein G antibodies (Invitrogen, 101223) were diluted (diluent mixture described above) by a factor of 4000, HRP conjugated IgM antibodies (ThermoFisher, A18835) were diluted by a factor of 2000, HRP conjugated IgA antibodies (ThermoFisher, PAI-74395) were diluted by a factor of 8000, HRP conjugated IgG1 antibodies (Invitrogen, MH1715) were diluted by a factor of 1000, HRP conjugated IgG3 antibodies (Invitrogen, 05-3620) were diluted by a factor of 1000, and HRP conjugated IgG4 antibodies (ThermoFisher, MH1742) were diluted by a factor of 2000 (Table 1). IgG1suggested: (Thermo Fisher Scientific Cat# MH1742, RRID:AB_2539714)HRP conjugated IgG3suggested: (Innovative Research Cat# 053620, RRID:AB_1500903)HRP conjugated IgG4suggested: NoneA set of 92 (used for each SARS-CoV-2 spike antibody ELISA) or 82 (used for total IgG RBD) negative samples from a pre-COVID-19 biorepository and four positive SARS-CoV-2 convalescent samples (2 positives for IgG4) were used in determining specificity of our ELISA methodology. total IgGsuggested: NoneIgG4suggested: NoneExperimental Models: Cell Lines Sentences Resources 2.3 Neutralization Methodologies: Cell lines and growth conditions: Vero E6 and HEK293T cells were obtained from ATCC and HEK293T cells stably transfected with human Angiotensin-Converting Enzyme 2 (HEK293T-hACE2) were obtained from BEI Resources (NR-52511). HEK293Tsuggested: NoneWells were washed twice with sterile water, and HEK293T-hACE2 cells were plated at 2.5×104 cells/well in 50 µL cDMEM. HEK293T-hACE2suggested: RRID:CVCL_A7UK)The setup plate was incubated at 37°C + 5% CO2 for 1 hour prior to addition to HEK293T-hACEs cell as above. HEK293T-hACEssuggested: NonePRNT assay: One day prior to infection, Vero E6 cells were plated at 4 × 105 cells/well in 2 mL cDMEM in a 6-well plate. Vero E6suggested: RRID:CVCL_A7UJ)Software and Algorithms Sentences Resources These plate cutoff values were then used to calculate each sample EPT using a 4-parameter logistic model in XL-fit software (model 208) as a Microsoft Excel add-in, as used by our team in previous clinical trial evaluations39,40. Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)Pseudovirus inhibition curves and the concentration of plasma required to inhibit pseudovirus entry by 50% (IC50) was determined by plotting the data and fitting a curve using the neutcurve Python package (https://jbloomlab.github.io/neutcurve/). Pythonsuggested: (IPython, RRID:SCR_001658)Results from OddPub: Thank you for sharing your data.
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.
Results from scite Reference Check: We found no unreliable references.
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