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Abstract

Aim Renal allograft failure accounts for nearly 30 percent of patients awaiting renal transplantation. Our aim was to determine if key proteins associated with fibrosis are targets for immune response leading to allograft rejection and may have promise as diagnostic and/or prognostic biomarkers. Methods Type II epithelial-to-mesenchymal transition (EMT) is reported to be involved in renal interstitial fibrosis. Five custom ‘direct-capture’ assays were developed on the Luminex immunobead platform for putative EMT autoantigens, including vimentin, beta catenin, p53, phosphoglycerate mutase and survivin. Extensive analytical validation for both single- and multi-plex assays was performed to establish the standard test performance characteristics. The diagnostic value of these assays were evaluated using diagnostic serum samples from cases of chronic renal allograph rejection (n = 25) and age-matched controls (n = 21). All measurements were performed on a Luminex FlexMAP 3D system equipped with xPONENT v3.2 and data analyzed using IBM SPSS v19.0. Results All analytical validations showed excellent accuracy and inter- and intra-batch precision across wide ranges of quantitation (1.5 ng/mL – 4 μ g/mL). Cross reactivity of P53and PGAM within the 5-plex assay necessitated a 3- and 2- plex assay configuration to resolve these issues. Validated multiplexed assays were then used to establish background levels of autoantibodies in an age-matched cohort prior to evaluation of autoantibody titre in the cases of chronic allograph rejection. The Mann-Whitney U test found that autoantibodies against P53 and vimentin were highly significant (p Conclusions Our findings support the idea that type II EMT may be highly associated with autoimmunity in chronic allograph rejection.