2.4. T-Cell ELISPOT

T-cell responses were measured essentially as described [14,19]. Briefly, ELISPOT plates were coated with capture antibody specific for the cytokine and species of interest (mouse, monkey, or rabbit IFNγ or IL-5; all obtained from commercial sources including MABTECH AB [Cincinnati, OH, USA] and Cell Sciences [Newbury Port, MA, USA]), incubated, washed, and blocked. Mouse splenic lymphocytes or monkey or rabbit PBMC were added at 2 × 10⁵ cells/well along with T1B or T1BT* peptide (0.1 mL/well of 5 μg/mL). Plates were incubated overnight at 37 °C and washed, and species-specific biotinylated anti-cytokine antibody was added (100 μL/well at 2 μg/mL in PBS + 1% BSA). Plates were incubated for one hour at room temperature, washed three times, and 100 μL/well of streptavidin-alkaline phosphatase (R&D Systems, Minneapolis, MN, USA) was added. Plates were incubated for one hour, then washed three times before the addition of 100 μL/well of BCIP/NBT substrate (R&D Systems). Plates were incubated for 15 min in the dark to allow the formation of spots, and the reaction was stopped by rinsing the wells with water. Plates were air-dried overnight, and the spots were counted on an AID Viruspot Reader (AID, Strassberg, Germany).