2.7. Analysis of T CD4+ IFN-γ Producing Cells in Animals Immunized with Hybrid Antibodies (ELISPOT)

For the ELISPOT assay, we used the “Ready-Set-Go” (eBioscience, San Diego, CA, USA) kit for the detection of IFN-γ and nitrocellulose plates with 96 wells (MAIPS 4510, Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The day before splenocyte isolation, the capture antibody was diluted in sterile buffer that was specific for sensitization of the PVDF membrane of the ELISPOT plate. After the addition of 100 mL per well of diluted antibody, the plate was incubated at 4 °C for 16–18 h and then blocked by using RPMI supplemented with 10% sterile FBS for 1 h at room temperature. Splenocytes were plated at 3 × 10⁵ cells per well, and medium containing rIL-2 at a final concentration of 30 U/mL and peptide P10 at a concentration of 20 mg/mL were added to their respective wells. A medium containing rIL-2 and 2.5 mg/mL Concanavalin A was used as a positive control. As a negative control, only rIL-2 was used, and the cells were stimulated in vitro with an unrelated peptide (Tewet). Cells were incubated in a CO₂ incubator at 37 °C for 24 h. To remove any residual cells, the plates were washed 3 times with PBS containing 0.05% Tween 20. A detection antibody was added for 2 h at room temperature, the plate was washed again, and streptavidin HRP × 250 was added. After 45 min, the plate was washed and developed by using AEC substrate (Becton Dickinson, Franklin Lakes, NJ, USA) for approximately 30 min. The plates were then dried at room temperature, and spots were counted in an automated counter (AID GmbH, Strassberg, Germany).