Isolation and Immortalization of Antigen-Specific Memory B Cells

IgM or IgG memory B cells were isolated from frozen peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting with 0.5 μg ml⁻¹ anti-CD19-PECy7 antibodies (BD, 341113) and mouse anti-PE microbeads (Miltenyi Biotec, 130-048-081), followed by FACS sorting using 3.75 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG (Jackson ImmunoResearch, 109-606-170), 5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgM (Invitrogen, A21215) and 1/40 PE-labeled anti-human IgD (BD, 555779). As previously described^{47 (link)}, sorted B cells were immortalized with Epstein-Barr virus (EBV) and plated in single cell cultures in the presence of CpG-DNA (2.5 μg ml⁻¹) and irradiated PBMC-feeder cells. Two weeks post-immortalization, the culture supernatants were tested (at a 2/5 dilution) for binding to PfSPZ by flow cytometry using a no-wash protocol. Briefly, cryopreserved PfSPZ were thawed, stained with the supernatants in 6.25× SYBR Green I for 30 min at room temperature, and incubated with 2.5 μg ml⁻¹ Alexa Fluor 647-conjugated goat anti-human IgG or anti-human IgM for 1 hour at 4°C. Only supernatants that did not bind to control beads were selected to exclude polyreactive antibodies.