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Abstract: There is currently a significant interest in the identification and validation of HLA-restricted CTL epitopes, which are thought to have important implications for the development of preventive and/or therapeutic applications in bacterial or viral infections, autoimmune diseases, and cancer. To better facilitate epitope discovery and validation, we present a cell- and radioisotope-free HLA-A⁎0201 assay system which relies upon fluorescence polarization. The assay has the advantage of allowing real-time measurements in solution without separation steps. In this report, we directed our efforts towards enhancing the sensitivity and reproducibility of the assay by conducting an in-depth analysis of parameters critical for standardization. Initial experiments demonstrated that the attachment of a fluorescence moiety at positions 5 and 8 for 9-mers and positions 5 and 6 for 10-mers, respectively, does not interfere with ligand binding to soluble HLA-A⁎0201. In addition, it was found that their binding to HLA-A⁎0201 was very effective showing high affinity binding with K d''s between 10.7 to 21.8 nM and binding capacities of up to 37%. In order to deliver maximized responses, factors such as the regulation of thermal HLA activation parameters to initiate peptide exchange as well as the specific adjustment of assay components were identified. Overall, the results obtained clearly demonstrate high accuracy, sensitivity and reproducibility of the FP-based assay approach. With the need for both increased throughput and miniaturized volumes, this fully homogenous, fluorescent-type binding assay is expected to be useful for routine analysis of peptide binding to MHC class I as well as class II molecules. [Copyright &y& Elsevier]