Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

An efficient method to make human monoclonal antibodies from memory B cells

This result represent one of the most important achievements within the MEMOVAX project. Infact, we have developed an improved method of Epstein-Barr virus transformation of human B cells. We have used this method to analyse the memory repertoire of a patient who recovered from severe acute respiratory syndrome coronavirus (SARS-CoV) infection and and to isolate monoclonal antibodies specific for different viral proteins. The technology is hereafter described.

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 9 U-bottom microplates in complete medium containing 2.5 g/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). Out of 56 attempts, 43 (76%) led to the isolation of one or more clones producing antibodies of the selected specificity. The EBV clones were stable, and monoclonal antibodies were recovered in the culture supernatants at a concentrations of 3-20 µg/ml.

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Via Vincenzo Vela 6
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