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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: The apparent clinical and research utility of soluble HLA (sHLA) underscores the need to further investigate immunological effects triggered by injections into mice. Developing reliable methods for immune response monitoring is crucial for individualizing therapies aimed at extending organ survival and improving patient health. In this study, we examined the humoral immune response elicited by immunization of sHLA into mice. Methods: The method involved five steps: (1) production/purification of injectable grade sHLA; (2) immunization of 9 female BALB/c mice with 25, 50, and 150 μg of sHLA-A*02:01; (4) injections at day 0, 21, 42, 63; (5) blood samples taken at day 0, 21, 42, 63, 72; (3) monitoring immunized mice for HLA-specific antibody production by an in-house, 120-allele Luminex-based single specificity solid phase assay. Results: Our results indicated that all mice immunized with sHLA-A*02:01 triggered an immune response. Titration analysis allowed appropriate measurements of semi-quantitative relationships between samples at different stages. It was noted that mice with 150 μg injection doses were capable to elicit high antibody titers as early as 21 days. Mice at lower injection doses had a slower time course. The average immune response distribution between loci was found to be highest in A alleles followed by C and weakest in B alleles. Overall, data indicate the generation of antibodies of broad specificity rather than against allogeneic determinants. Conclusions: Low variability between Balb/c mice is explained by the fact that all mice have an identical genetic makeup. These findings underscore the potential importance of polymorphisms in the MHC locus on immune responses. Furthermore, this data may allow increasing accuracy and efficiency in the determination of immunological responses that could help monitor occurrences observed in humans. In addition, injectable sHLA may serve as quality reagents in the creation of therapeutic/diagnostic anti-HLA antibodies or to induce tolerance. [Copyright &y& Elsevier]

Aim: Therapies exist for the bulk removal of antibodies in sensitized transplant patients, but there are no means to specifically deplete antibodies that recognize a specific HLA molecule. Current bulk antibody removal procedures may leave a patient compromised immunologically and more susceptible to opportunistic infections. The goal of this project was to create specific HLA-A∗02:01 removal matrices and demonstrate their ability to capture defined anti-HLA antibodies. Methods: Soluble class I HLA-A∗02:01 molecules were produced in a native conformation in mammalian cells, purified by affinity chromatography, coupled to a Sepharose matrix, and loaded into a column enclosure. Various columns were created and tested with an automated chromatography system by initial injection of various HLA antibodies under different loading conditions. After an extensive column wash, the bound antibodies were released by a pH shift and quantitated. Loading and elution profiles were recorded. Results: Initial tests show that HLA columns maintain their structural integrity and function. Multiple passes of the antibody W6/32, recognizing only intact HLA molecules, resulted in consistent and repeatable binding patterns. During the entire performance process, several parameters were identified, determining column saturation, capacity and efficiency values. Specificity was shown by removing a typical A∗02:01 recognition pattern from an A2 sera. In addition, treatment conditions were established to reuse the device. Conclusions: Anti-HLA antibody removal devices are highly efficient in selectively depleting HLA antibodies. Capable of removing up to 1 mg per ml of matrix depending on the avidity of the antibody makes this device very efficient for research studies of HLA antibodies and provides promise for therapeutic applications. Furthermore, this device might also find an application in HLA diagnostics, allowing for the deconvolution of serologic samples and providing a new way to evaluate high PRA patients. [Copyright &y& Elsevier]

Aim: Many sensitized transplant patients have HLA-specific antibodies due to previous graft failures, blood transfusions, and pregnancies. These humoral responses are typically polyclonal in nature, antibody composition is distinct to each individual, and it is difficult to precisely determine antibody specificity. Distinct antibodies cannot be readily isolated for individual characterization. In this study we isolate distinct anti-HLA antibodies to evaluate their phenotypic and functional traits. Methods: After characterizing sera from highly sensitized patients with a single antigen assay, sera were passed over HLA-specific separation devices made by coupling purified soluble HLA to a sepharose matrix. A scheme of multiple separation rounds involving loading and elution cycles with different HLA allele combinations was applied. This process resulted in mono-specific patterns of HLA reactivity measured by a Luminex-based bead assay. Results: Our results show that complex patterns of sera reactivity can be reduced to more simplified outputs allowing better interpretation of individual antibody populations. Separation of IgG, IgA, and IgM further assisted in the interpretation of these responses. Furthermore, this technique was also of use in evaluating false positive/negative assay signals, strengthening otherwise weak antibody reactivity, and eliminating background interference. Conclusions: Antibody concentration, isotype, epitope specificity, cross-reactivity, and the ability to fix complement have all been implicated as factors that contribute to the pathogenicity of anti-HLA antibodies following organ transplantation. The deconvolution of antibody reactivity patterns can assist in the assessment of risk factors for immunologic rejection as well as provide new insights into anti-HLA antibody profiling and epitope structure analysis. [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: Solid phase antibody detection assays are used in most clinical histocompatibility laboratories. Recent studies have shown that current methodologies can be inconsistent within and between laboratories. Understanding the source of assay inconsistency will promote standardization. In this study, we investigated secondary antibody systems and HLA integrity inconsistencies to facilitate a standardization strategy for HLA antibody detection assays. Methods: A soluble HLA-based assay was tested in a three step protocol; (1) binding of soluble HLA to a solid support, (2) incubation of the coupled HLA with sera/antibody, and (3) visualization of anti-HLA antibodies using a detection system. Nearly 200 soluble HLA molecules provided the platform content and more than 40 positive and negative controls were included to address Ig isotype specificity, HLA structural integrity on the platform, capacity of the support, and additional contributing factors. Results: Consistency challenges in anti-HLA antibody testing are multi-layered. In the secondary antibody detection system, anti-IgG PE antibodies crossreact with IgM controls resulting in non-proportional signal amplification because of IgM’s pentameric nature. Furthermore, variation in the avidity of tested secondary antibodies influences a consensus MFI. Commercial bead systems compared to soluble HLA revealed variation in bead HLA content. Structural disintegration of particular HLA impaired bead capacity by two logs, altered MFI saturation, and a linear detection range could only be achieved by testing various dilutions of sera samples. Conclusions: First, identifying assay inconsistencies and then standardizing methods, reagents, and controls to eliminate these inconsistencies will achieve concordance within and between laboratories. Here, we characterize variability in secondary antibodies, bead specificities, and HLA integrity on bead preparations. Having characterized these assay variables, options for assay standardization are discussed. [Copyright &y& Elsevier]

Aim: Solid phase antibody detection assays using single antigens represent a cutting edge technology for screening the sera of transplant patients. Considering the large number of HLA targets, it has been difficult to QC all single allelic HLA entities with well characterized serological reagents. Our aim is to identify, generate, and characterize agreeable sera/antibodies for the quality control of single HLA antigens produced by various manufacturers. Methods: Monospecific antibodies are considered the most appropriate means for the quality control of single antigens. A number of human and mouse monoclonal antibodies represent excellent reagents for quality assessment. However, monoclonal antibodies are not available for many HLA. Here, deconvolution of serological specimens, isolating mono-specific anti-HLA antibodies by an absorption-elution technology further assisted in increasing the reagent pool. We then utilize these monospecific antibodies for single HLA antigen QC purposes. Results: We characterized HLA monospecific antibodies from dozens of candidate reagents. The purified antibodies were highly specific for specific HLA targets. Initial titration curves indicate an optimal performance range with a specific HLA. Thereafter, each monospecific reagent was screened for its HLA cross-reactive pattern. Crossreactivity coincides with known epitopes and suggests that clinically relevant and irrelevant epitopes are indicated. Conclusions: Well-characterized sera/antibodies provide not only a consensus for antibody specificity, helping to standardize and QC existing HLA platforms, but also allow a consensus for the strength (MFI) of the antibody-HLA interaction. The provision of multiple HLA monospecific antibodies that can be harvested from patient sera by individual laboratories will enable multiple HLA single antigen QC. Furthermore, harvest of HLA monospecific sera will provide insights into HLA epitope structures, supporting the efforts to understand crossreactive HLA in the clinical lab. [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]