Lectin pokeweed mitogen

MBC stimulations were performed on peripheral blood mononuclear cells (PBMCs) collected from subjects in the convalescent cohort. To induce MBC differentiation into antibody secreting cells, 1×10⁶ PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 μg/ml CpG (Invitrogen) in complete RPMI in an incubator at 37°C/5% CO₂ for 5 days. After stimulation, cells were counted and added to ELISpot white polysterene plates (Thermo Fisher) coated with 4 μg/ml of SARS-CoV-2 spike that were blocked with 200 μl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37μC/5% CO₂. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti-IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature. Plates were washed and developed with NBT/BCIP (Thermo Fisher Scientific) for 2–10 minutes, and reactions were stopped by washing plates with distilled water and allowed to dry overnight before counting. Images were captured with Immunocapture 6.4 software (Cellular Technology Ltd.), and spots were manually counted.