Detection of Antigen-Specific Memory B Cells

PBMCs were pre‐activated with TLR agonist R848 (1 μg ml⁻¹, InvivoGen) and human IL‐2 (100 unit per ml, PeproTech) for 3 days. The activated B cells were washed and performed ELISPOT assay as previously described (Wu et al., 2018 (link)). Briefly, MultiScreen 96‐well plates were coated with PAM trimer (1 μg per well) or anti‐monkey IgG antibodies (1 μg per well). 2 × 10⁵ cells were seeded for the detection of PAM trimer‐specific IgG‐secreting cells, or 5 × 10⁴ cells for the detection of total IgG‐secreting cells. After overnight incubation, cells were lysed and probed with HRP‐conjugated rabbit anti‐monkey IgG (H&L). Spots were developed using 3‐amino‐9‐ethylcarbazole (AEC) substrate (BD Pharmigen). Spots of antibody‐secreting cells (ASC) were counted using an ELISPOT reader and data are presented as the number of memory B cells per million cells.