HEK293T cells were transiently transfected using the PEI method with the SARS‐CoV‐2‐spike‐encoding plasmid pCG1_CoV_2019S [41 (link)] in combination with a GFP encoding plasmid. HEK293T cells transfected only with a GFP‐encoding plasmid served as a negative control. Two days after transfection, SARS‐CoV‐2 spike/GFP‐co‐transfected and GFP‐only transfected HEK293T cells were stained with serum samples (1:100 dilution) from SARS‐CoV‐2‐infected patients and noninfected controls followed by staining with a secondary antibody mixture (2° antibody mix) consisting of PE‐conjugated anti‐human IgA (Southern Biotech, Birmingham, AL), AF647‐conjugated anti‐human IgG (Southern Biotech), and DyLight405‐conjugated anti‐human IgM (Jackson Immuno Research, Dianova, Hamburg) antibodies. Cells were analyzed using a Gallios flow cytometer (Beckman Coulter) or a Cytoflex flow cytometer (Beckman Coulter) and Kaluza flow cytometry software (Beckman Coulter).
Protocol detail
SARS-CoV-2 Spike Protein Antibody Detection
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PE‐conjugated anti‐human IgA
DyLight405‐conjugated anti‐human IgM
Gallios flow cytometer
Cytoflex flow cytometer
Kaluza flow cytometry software
Af647
Anti iga
Anti igg
Anti igm
Antibodies
Antibody
Cells
Dilution
Flow cytometry
Human
Patients
Plasmid
Sars cov 2
Serum
Transfection
Corresponding Organization : Friedrich-Alexander-Universität Erlangen-Nürnberg
Other organizations : German Primate Center, University of Göttingen, University of Freiburg
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Variable analysis
independent variables
- Transfection of HEK293T cells with SARS-CoV-2 spike-encoding plasmid pCG1_CoV_2019S in combination with a GFP-encoding plasmid
- Binding of serum samples (1:100 dilution) from SARS-CoV-2-infected patients and non-infected controls to SARS-CoV-2 spike/GFP-co-transfected and GFP-only transfected HEK293T cells
- Staining of bound serum samples with a secondary antibody mixture consisting of PE-conjugated anti-human IgA, AF647-conjugated anti-human IgG, and DyLight405-conjugated anti-human IgM antibodies
- Analysis of stained cells using a Gallios flow cytometer or a Cytoflex flow cytometer and Kaluza flow cytometry software
- HEK293T cells transfected only with a GFP-encoding plasmid
- Serum samples from SARS-CoV-2-infected patients
- Serum samples from non-infected controls
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