After informed consent was obtained, peripheral blood was collected from a 35-year-old patient who had recovered from infection by SARS-CoV. IgG+ memory B cells were isolated by binding to CD22 microbeads (Miltenyi) followed by depletion of cells carrying IgM, IgD and IgA by cell sorting. Memory B cells were seeded at 10 or 50 cells per well in 96 U-bottom microplates in complete medium containing 2.5 μg/ml CpG 2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well). After 2 weeks, the culture supernatants were screened for specific antibodies. Positive cultures were cloned by limiting dilution in the presence of CpG 2006 and irradiated mononuclear cells. Antibody was purified from culture supernatants by affinity chromatography on protein A columns (Amersham).
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Isolation of SARS-CoV Memory B Cells
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Affinity chromatography
Allogeneic cells
Antibodies
Antibody
Blood
Cells
Cpg 2006
Dilution
Infection
Memory b cells
Microbeads
Patient
Protein a
Sars cov
Corresponding Organization :
Other organizations : Institute of Oncology Research, National Institutes of Health, Newron Pharmaceuticals (Italy), Luigi Sacco Hospital
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Variable analysis
independent variables
- Seeding of memory B cells at 10 or 50 cells per well
- Presence of EBV (30% supernatant of B95-8 cells)
- Presence of irradiated allogeneic mononuclear cells (50,000 per well)
- Specific antibody production in culture supernatants
- Antibody purification from culture supernatants by affinity chromatography on protein A columns
- Presence of CpG 2006 (2.5 μg/ml) in the complete medium
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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