Quantifying H1N1-Specific Memory B Cells

H1N1-specific memory B cells (MBC) were measured from T2 by ELISPOT assay as described^{14 (link)}. For MBC enumeration, previously cryopreserved PBMC were thawed, rested, and stimulated with 5 µg/mL H1N1/09 vaccine antigen plus anti-CD28 mAb (1 µg/mL), CpG (as a positive control) or unstimulated (as negative control) for 5 days at 37 °C. On day 5, cells were plated in wells coated with goat anti-human IgG (2 µg/mL, Jackson Immunoresearch) at 100,000 cells/well for 4 hours at 37 °C and assayed for H1N1-specific IgG. Spots were read using Immunospot software and plate reader (Cellular Technologies LTD). H1N1-specific IgG results were normalized and graphed as proportion of the positive control.