CPT tubes will be centrifuged at 1800g (approximately 2800 rpm on a Sorvall RT6000 centrifuge) for 20 min. at room tempe rature. After centrifugation, plasma in the CPT tubes will be gently pipetted against the gel plug to dislodge cells stuck to the top of the gel. The cell suspension will be transferred to a 50 mL conical polypropylene tube. cRPMI will be added to a total of 40 mL. A 10 mL aliquot of cell suspension for counting will be removed. The 50 mL tubes will then be centrifuged at 250g for seven minutes at room temperature. When centrifugation is complete, the supernatant will be aspirated. PMBCs will be either cryopreserved or used fresh. 9.2 Immune Assays 9.2.1 Cellular immune response assays – Elispot Antigen-specific memory T cell responses are assayed by means of a IFN  ELISPOT a ssay. PVDF ELISPOT (enzyme-linked immunoSPOT) plates (Millipore) are coated with an a nti-IFN monoclonal antibody (mAb) (clone D1K, MabTech) overnight, after whic h time plates are blocked and washed with phosphate-buffered saline/Tween (PBST). Cryopreserved PBMC, thawed and rested overnight in medium are then plated in t riplicate at 3x105 PBMC/well in a total volume of200 L/well with either medium alone or with medium containing antigen (PA2024: PAP-GMCSF fusion protein, PAP or PHA – Phytohaemagglutinin, an assay control). Plates are then incubated for 40-48 hrs a fter which time plates are washed then incubated with Strepatavidin conjugated anti- Page 31 of 50 IRB Protocol No.