Immortalization of gp120-specific Memory B Cells

The cryopreserved PBMCs from Subject 009 were thawed and cultured in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 1% L-glutamine and 1% Penicillin-Streptomycin (P/S) at 37°C with 5% CO2 overnight. The B cells from overnighted culture PBMCs were first enriched using MACS Human B Cell Isolation Kit II (Milteny Biotec, San Diego, CA). Then, CD27+ memory B cells were isolated using human memory B cell isolation kit (Milteny Biotec). For EBV immortalization of B cells, the memory B cells isolated from Subject 009 were seeded at 5 cells/well in 96-well U-bottom microplates in 200µl of complete RPMI medium containing 2.5µg/ml CpG ODN2006, in the presence of EBV (30% supernatant of B95-8 cells) and irradiated allogeneic mononuclear cells (50,000 per well) ^{48 (link)}. After 12 days of culture at 37°C with 5% CO2, 50µl of culture supernatants were harvested and screened for gp120 binding by ELISA. The EBV transformed cells from the gp120 binding positive wells were picked and expanded for clonal culture. The antibody from positive wells were further determined by ELISA with anti-Lamda and anti-Kappa secondary antibody, separately. The cells from gp120-specific clone #64 were harvested and preserved in RNALater (Qiagen, Redwood City, CA) for RNA extraction and Ig gene cloning.