Background/Purpose: Serum 14-3-3 eta, a protein biomarker that is differentially expressed in rheumatoid arthritis (RA), has been reported to add incrementally to rheumatoid factor (RF) and anti-cyclic citrullinated protein (anti-CCP) in diagnosing both early and established RA. We recently reported that extracellular 14-3-3 eta behaves as a novel mediator of RA pathogenesis, inducing factors involved in driving both inflammation (TNFα) and joint re-modeling (MMP-9) and that serum 14-3-3 eta is an independent predictor of damage in patients with RA and psoriatic arthritis (PsA). Since 14-3-3 eta is normally an intracellular chaperone that is externalized through an exosomal-mediated process by a disease-specific trigger, we investigated whether its extracellular presence elicits an autoantibody response that may be measured for clinical utility.

Methods: Detection of 14-3-3 eta autoantibodies was evaluated by examining immunoreactivity towards human recombinant 14-3-3 eta by immunoblot analysis using serum from healthy subjects and those with RA. Epitope mapping and in silico modeling was performed to identify putative epitopes of which 11 peptide regions were selected. To further specify high-priority regions, the serum expression levels of 14-3-3 eta autoantibodies were evaluated in 30 healthy individuals and 58 RA patients. Individual sequences were selected based on their absolute specificity for RA, of which 4 of 11 met this criteria with sensitivities ranging from 34 – 62%. A composite score was generated by combining the fluorescence intensity (FI) measurements for these 4 peptides, each of which were equally weighted and expressed in units (U). T-tests and Mann-Whitney U-tests were used to determine group differences. The area under the ROC curve (AUC) was generated for diagnostic utility estimates and likelihood ratios (LR) for various 14-3-3 eta autoantibody composite cut-offs.

Results: Immunoblot analysis reveals that patients with RA possess autoantibodies directed towards 14-3-3 eta. This prototype 14-3-3 eta autoantibody assay exhibits high specificity for RA versus healthy controls with means (SD) and medians (min-max) of 828U (1292U) and 681U (340U-10386U) for RA and 385U (74U) and 371U (289U-568U) for healthy controls, p-value <0.0001. The ROC AUC was 0.93 (95% CI 0.88-0.98; p<0.0001). At the best cut-off of 476U, the specificity and sensitivity were 90% and 80%, respectively with a likelihood ratio positive of 7.9 increasing to 18 at 580U. These prioritized sequences will be further specified based on their differential expression in RA versus disease controls to develop an anti-14-3-3 eta ELISA for clinical diagnosis. 

Conclusion: 14-3-3 eta is an RA biomarker whose extracellular expression elicits an autoantibody response that is highly specific for RA compared to healthy individuals.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-autoantibodies-to-14-3-3-eta-are-highly-specific-for-rheumatoid-arthritis/