Identification of T-cell epitopes using ELISpot and peptide pool arrays

Here we describe a method for T-cell epitope identification using a modified ELISpot assay that is both simple and efficient. By using a carefully constructed array of pools of overlapping peptides spanning the entire antigen sequence to stimulate T-cell responses, we are able to detect antigen-specific cytokine responses by both CD8+ and CD4+ T cells and identify the specific peptides to which the cells are responding. Additionally, by performing magnetic bead depletion of either CD8+ or CD4+ cells prior to the assay, we are able to determine the phenotype of the responding cells to each of the peptide epitopes identified. Use of this method will allow the identification of both CD4+ and CD8+ T-cell epitopes without the need for MHC allele-matched reagents and without the need for highly specialized instrumentation. By using an array of peptide pools, this method also dramatically reduces the number of immune cells required to test the entire antigen sequence, often a limiting factor in vaccine testing and other studies.