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Aim: Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. Methods: A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Results: Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Conclusions: Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses. [Copyright &y& Elsevier]

Aim: Degraded or denatured HLA proteins can contribute to false positive, false negative, and inconsistent MFI signals. To provide recommendations for improvements in the manufacturing process, HLA protein degradation was systematically studied from the perspective of physical instability; changes in higher order structure following acid and base hydrolysis, thermal degradation, reagent changes, and freeze-thaw cycles were analyzed. The goal was to identify optimal physical conditions for maintaining HLA integrity. Methods: To detect significant changes in the quality and function of soluble HLA, specific bead sets loaded with different allele specificities were created and tested under stress conditions using the Luminex platform. Various monoclonal antibodies as well as sera specimen were used to probe the effects of pH variations and temperature ranges, specific chemical agents, or working conditions. Results: These HLA stress studies showed that the exposure of soluble HLA loaded beads tolerated temperatures up to 50°C with no detectable loss of activity. Freezing did cause a reduction in activity that could be overcome by using stabilizers such as glycerol. Favorable results were also found while investigating the pH range, with optimal performance up to pH 10.5; a basic environment is preferred. Acidic environments down to pH 5.5 were accepted, but bead activity declined below pH 5.5. Conclusions: Stress testing can identify optimal conditions and prevent deleterious conditions. Here, a number of analytical techniques characterized alterations in HLA protein primary structure, physical stability, and activity. Our results indicate optimal conditions, propose stability-indicating methodologies, and provide a means for testing HLA protein activity and potency. The question of how much degradation is acceptable is still pending – we found that a level above 90% intact HLA is preferred. Overall, this study provides valuable information to improve future formulations and manufacturing processes. [Copyright &y& Elsevier]

Aim: While it is generally accepted that HLA antibodies represent significant risk factors for transplant rejection and graft failure, it has become apparent that current methodologies are not delivering all the answers to accurately assess transplant patients. Over the years, many controversies and shortcomings have been discussed concerning solid phase antibody detection assays but only a few improvements have been implemented. To better accommodate the increasing demand for more accurate and meaningful data, we set out to develop a new generation of solid phase single antigen assays using soluble HLA. Methods: A single specificity solid phase assay platform was created utilizing 120 soluble HLA alleles and a large panel of control proteins, all of which were validated by qualified monoclonal antibodies. The assays were optimized in regard to sera incubations, wash steps and PE-detection. In some cases, serological specimens were treated using supplemental techniques to reduce sera complexity or remove interference and background problems. Results: In several case studies, more distinctive reaction patterns compared to other assay manufacturers could be demonstrated. The insight into isotype distribution provided helpful facts to draw further conclusions regarding the patient’s immunological state. The usage of soluble HLA demonstrated a higher capacity in sera titration experiments while lack of substantial structural disintegration reduced the false positive rate allowing better definition of single cross-reactive patterns. Conclusions: The complex nature of human sera has set new challenges in the evaluation of the phenotypic and functional traits of antibodies. A large diverse panel of highly purified functionally intact HLA antigens along with a definitive set of control proteins is essential to satisfy the increasing demand for meaningful immunological assessment of transplant patients. [Copyright &y& Elsevier]