Enzyme-linked immunospot (ELISPOT) assay for IFN-γ
Since CTLs are known to produce the Th1 cytokine IFN-γ, peptide-specific T cells were enumerated by measuring IFN-γ-producing cells by ELISPOT assay. As shown in Fig. 2, neuritin13–21, neuritin121–129 and neuritin4–12 peptides were found to generate a strong peptide-specific T cell response by virtue of their ability to induce increased frequencies of IFN-γ-producing T cells, as compared to the negative peptide control. These results suggest that neuritin peptide vaccines can increase IFN-γ secretion by effectors and enhance the Th1 immune response.
Fig. 2 Specific IFN-γ by ELISPOT assay. The PBMCs of human HLA-A2+ donors were obtained and then cultured in RPMI 1640 supplemented with 10 % FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Dendritic cell were generated, and loaded with different peptides at a final concentration of 100 μg/ml for 4 h and were then irradiated with 20 Gy, which prevented all outgrowths in the control cultures. Autologous T cells were restimulated every 7 days with the peptide-pulsed DCs to generate peptide-specific CTLs. The IFN-γ secretion was then assessed on day 23. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05