Critical factors in the development of fluorescence polarization-based peptide binding assays: an equilibrium study monitoring specific peptide binding to soluble HLA-A*0201.

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.

Development and validation of a fluorescence polarization-based competitive peptide-binding assay for HLA-A*0201--a new tool for epitope discovery.

Various approaches are currently proposed to successfully develop therapies for the prevention and treatment of infectious diseases and cancer. One of the most promising approaches is the development of vaccines that elicit cytotoxic T lymphocyte (CTL) responses. Consequently, identification and exact definition of molecular parameters involved in peptide-MHC class-I interactions of putative CTL epitopes are of prime importance for the development of immunomodulating compounds. To better facilitate epitope discovery, we developed and validated a novel state-of-the-art biochemical HLA-A0201 assay, which is comprised of technologically advanced cutting edge reagents. The technique is based on competition and uses a FITC-labeled reference peptide and highly purified soluble HLA-A0201 molecules to quantitatively measure the binding capacity of nonlabeled peptide candidates. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. During standardization, the problem of assay parameter variation is discussed, showing the dramatic influence of HLA and reference peptide concentrations as well as the choice of the reference peptide itself on IC(50) determinations. For validation, a panel of 15 well-defined HLA-A0201 ligands from various sources covering a broad range of binding affinities was tested. Binding data were used to compare against pre-existing quantitative assay systems. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with recombinant sHLA molecules is highly advantageous for the accurate assessment of peptide binding. Furthermore, the assay also features high-throughput screening capacity, providing uniquely efficient means of identifying and evaluating immune target molecules.

Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization.

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.

Toward a definition of self: proteomic evaluation of the class I peptide repertoire.

MHC class I molecules present host- and pathogen-derived peptides for immune surveillance. Much attention is given to the search for viral and tumor nonself peptide epitopes, yet the question remains, "What is self?" Analyses of Edman motifs and of small sets of individual peptides suggest that the class I self repertoire consists of thousands of different peptides. However, there exists no systematic characterization of this self-peptide backdrop, causing the definition of class I-presented self to remain largely hypothetical. To better understand the breadth and nature of self proteins sampled by class I HLA, we sequenced >200 endogenously loaded HLA-B*1801 peptides from a human B cell line. Peptide-source proteins, ranging from actin-related protein 6 to zinc finger protein 147, possessed an assortment of biological and molecular functions. Major categories included binding proteins, catalytic proteins, and proteins involved in cell metabolism, growth, and maintenance. Genetically, peptides encoded by all chromosomes were presented. Statistical comparison of proteins presented by class I vs the human proteome provides empiric evidence that the range of proteins sampled by class I is relatively unbiased, with the exception of RNA-binding proteins that are over-represented in the class I peptide repertoire. These data show that, in this cell line, class I-presented self peptides represent a comprehensive and balanced summary of the proteomic content of the cell. Importantly, virus- and tumor-induced changes in virtually any cellular compartment or to any chromosome can be expected to be presented by class I molecules for immune recognition.