Generation of TOSV-specific antibodies

PBMCs and plasma samples were obtained from a patient with meningitis, infected by TOSV, during the acute phase and one year after the infection. At the Institute for Research in Biomedicine (Bellinzona Switzerland), frozen PBMCs were thawed and immortalized using EBV in the presence of CpG, as previously described [26 (link)]. Briefly, IgG⁺ memory B cells were isolated using magnetic cell separators (MACS) and negative and positive selection kits (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, memory B cells were seeded at 10 or 50 cells per well in 96 U-bottom microplates (Greiner, ThermoFisher Scientific) 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 by Intellicyt Cytometer against TOSV-infected VERO cells. Specific TOSV antibodies were purified from culture supernatants by affinity chromatography on protein A columns (Amersham) and stored at −20 °C for further testing.