Cryopreserved PBMCs were thawed and rested overnight at 37°C for batch analysis. The cells were then plated in triplicate of 3.0×105 cells/well and incubated with recombinant PAP or PA2024 at 25 µg/mL, leucoagglutinin PHA-L at 10 µg/mL (Sigma, Cat # L2769) or without antigen for 48 hours at 37°C in MultiScreen Filter Plates (Millipore, Cat # S2EM004M99). Cells secreting interferon-γ (IFN-γ) were visualized by anti-human-IFN-γ enzyme-linked immunospot assay (ELISpot) (MABTECH, Cat # 3420-2A). Plates were scanned with an automated ELISpot plate reader (CTL-ImmunoSpot Analyzer). Spots were counted using CTL Immunospot V.5.0 analyzer software. Final counts of antigen-specific IFN-γ secreting cells were obtained by subtracting the number of spots counted in no-antigen control wells from test wells. Samples were accepted for inclusion in final analysis if positive control PHA wells had an average >100 spots/well, and negative control (no antigen) wells had <100 spots/well.
For the proliferation assays, the cells were plated in triplicate of 1.0×105 cells/well and incubated with recombinant PAP or PA2024 at 25 µg/mL, leucoagglutinin PHA-L at 3 µg/mL (Sigma, Cat# L2769) or without antigen for 120 hours at 37°C. 3H-thymidine 1 mCi was then added, and the cells were incubated for another 8 hours at 37°C. Cells were harvests and assessed on a Perkin Elmer MicroBeta Trilux.
For the proliferation assays, the cells were plated in triplicate of 1.0×105 cells/well and incubated with recombinant PAP or PA2024 at 25 µg/mL, leucoagglutinin PHA-L at 3 µg/mL (Sigma, Cat# L2769) or without antigen for 120 hours at 37°C. 3H-thymidine 1 mCi was then added, and the cells were incubated for another 8 hours at 37°C. Cells were harvests and assessed on a Perkin Elmer MicroBeta Trilux.