INCREASE THE SENSITIVITY OF ELISPOT DETECTION

ABSTRACTThe 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-CD8 + T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specificstimulation 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 -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 stagingŽ protocols for autoimmune as well as tumor and infectious diseases. Key words: autoimmunity, memory, monitoring, T cells, type 1 diabetes