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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]