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 challenges that demand immediate attention. 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 that complements the portfolio of SARS‐CoV‐2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. 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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