Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection.

The identification of parameters maximizing detection sensitivity in ELISpot assays is important to transfer this technology into the clinical setting for identifying rare Ag-specific CD8(+) T cells. We have therefore considered human IFN-gamma CD8(+) T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specific stimulation period. Two parameters were found to greatly enhance detection sensitivity (i.e., to specifically increase epitope-driven signal while keeping background noise to a minimum): use of human serum-free vs. serum-supplemented culture medium (2.4-fold median increase) and addition of low dose IL-7 (1.5-fold increase). Incorporating both of these parameters into the ELISpot procedure proved capable of greatly amplifying (35.1-fold increase) the low grade CD8(+) T cell responses directed against beta-cell epitopes of type 1 diabetes patients, as compared to a previously optimized procedure using human serum-supplemented medium and low dose IL-2. Implementation of this ELISpot procedure should expedite development of "immune staging" protocols for autoimmune as well as tumor and infectious diseases.