Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera
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Abstract
Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. Among these is the urgent need for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. For this, sensitive, specific and quantitative serological assays are required. Here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay which complements the portfolio of SARS-CoV-2 serological assays. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.
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SciScore for 10.1101/2020.06.15.152587: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: SARS-CoV-2 positive sera were collected from 29 PCR confirmed symptomatic COVID-19 inpatients (n=17) or outpatients (n=12) treated at the University Hospital Heidelberg under general informed consent (ethics votum no S-148/2020, University Hospital Heidelberg). 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 Next, cells were incubated with patient serum (prediluted 1:1 in 0,4% Triton-X100 in PBS; further dilution 1:50 in PBS if not stated otherwise) and anti-ds-RNA mouse monoclonal J2 antibody (Scicons, … SciScore for 10.1101/2020.06.15.152587: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: SARS-CoV-2 positive sera were collected from 29 PCR confirmed symptomatic COVID-19 inpatients (n=17) or outpatients (n=12) treated at the University Hospital Heidelberg under general informed consent (ethics votum no S-148/2020, University Hospital Heidelberg). 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 Next, cells were incubated with patient serum (prediluted 1:1 in 0,4% Triton-X100 in PBS; further dilution 1:50 in PBS if not stated otherwise) and anti-ds-RNA mouse monoclonal J2 antibody (Scicons, 1:4000) in PBS for 30 min at room temperature. anti-ds-RNAsuggested: NoneJ2suggested: NoneExperimental Models: Cell Lines Sentences Resources Only very few infected calls were detected in the case of hepatocyte-derived carcinoma cells (HUH-7), human embryonic kidney (HEK293T) and human alveolar basal epithelial (A549) cells (Fig. S1). HEK293Tsuggested: NoneA549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)Calu-3 cells grew in small clumps, often on top of each other which impacted our microscopy-based readout. Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)For serum screening by IF microscopy, VeroE6 cells were seeded at a density of 7,000 cells per well into a black-wall glass-bottom 96 well plates (Corning, Product Number 353219) or on glass coverslips placed in a 24-well plate. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)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 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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