Interleukin 2 (il 2)

Patient peripheral blood mononuclear cells (PBMC) were isolated by ficoll-paque density gradient centrifugation. A stimulation cocktail containing 1 µg/mL R848 (Mabtech, Nacka Strand, Sweden) and 10 µg/mL IL-2 (Mabtech) was added for 72 h at 37°C/5% CO₂ to activate antibody production by memory B cells. The frequency of total IgM and IgG-producing cells in PBMC was determined using IgM or IgG ELISpot assays. Briefly, anti-IgM or IgG capture antibodies (Mabtech) were incubated in high protein-binding PVDF membrane plates (Millipore, Billerica, MA) for 5 h at RT. Activated cell suspensions were seeded in duplicate at 5,000, 2,500 and 1,250 cells/well and incubated for 8 h at 37°C/5% CO₂. Detection was performed by incubating biotinylated anti-IgM (Mabtech) or anti-IgG (Mabtech) detection antibodies for 2 h at RT. Streptavadin-conjugated alkaline phosphatase (R&D, Minneapolis, MN) was added for 2 h at RT followed by incubation with BCIP/NBT substrate (R&D) for 30 min at RT. The reaction was stopped by rinsing wells with ultrapure water. In parallel experiments to assess MDA-specific antibody-secreting cells, ELISpot plates were coated with 62.5 µg/ml MDA-modified BSA for 5 h RT. Cell suspensions were added in duplicate at 50,000, 25,000 and 12,500 cells/well and incubated for 8 h at 37°C/5% CO₂. ELISpot detection was performed as for total IgM and IgG detection.