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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: 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: Single antigen beads (SAB) assays are routinely used to identify anti-HLA antibodies and the level of detection is commonly communicated as mean fluorescence intensity (MFI). Many factors may contribute to an MFI value, including antibody concentration and interference of non-IgG isotypes. Here we purified polyclonal anti-HLA antibodies from sensitized patients and tested the influence of antibody concentration and non-IgG isotypes on MFI. Methods: Using an HLA-DR11 affinity column, we purified anti-HLA-DR11 antibodies from 13 sensitized human sera. The purified anti-HLA antibodies were then tested on a SAB assay using a secondary that only recognizes IgG isotypes. IgG isotypes were separated from IgM and IgA isotypes via size exclusion HPLC. Reduction of multimeric antibodies (IgM, IgA) to monomers was preformed with 100mM DTT at 4°C overnight with a total antibody concentration at 1mg/ml. Results: Serial dilution experiments with affinity purified anti-HLA antibodies demonstrated a strong correlation between antibody concentration and MFI within a single patient. However, between patients, the MFI value did not correlate to an antibody concentration – MFI was not comparable between patients. When IgM and IgA multimers were separated from IgG, a significant increase in MFI was observed compared to the original mix of isotypes. Furthermore, reduction of IgM and IgA into monomers with IgG did not impact MFI values as the monomeric mix provided an MFI matching the IgG monomer only. Conclusions: Multiple factors influence SAB MFI. Here we tested the influence of both antibody concentration and the presence of IgM, and IgA on MFI. We show that MFI correlates with antibody concentration within a particular patient but not between patients. We also show that mixing IgM and IgA multimers (but not monomers) with IgG antibodies interferes with the assay by reducing MFI. Together these data exemplify the complexity of anti-HLA antibodies and factors that influence the SAB that is used to detect them. Cate: Pure Protein: Employee. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Osborn: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor. [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]