10-P

Abstract

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. 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. 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. 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.