Abstract
Background:The presence of autoantibodies in Rheumatoid Arthritis (RA) is a hallmark of the disease and one of main criteria for diagnosis and clinical classification. Identification of anti-citrullinated protein antibodies (ACPA) and RF-Factors in circulation of RA patients remains the most characteristic and established criteria for diagnosis. However, due to diagnostic limitation of these biomarkers, only approximately 70% of patients with RA can be identified. As a consequence, there is a lack of diagnostic biomarkers for large number of patients. Diagnosis of these patients relies mainly on assessment of their clinical symptoms such as swollen and tender joints. This problem leaves a need for diagnostic improvement and development of more sensitive biomarkers.Objectives:In this study, we aimed to develop a sensitive biomarker assay capable to identify and quantify presence of autoantibodies against denatured type II collagen in circulation of patients with RA.Methods:The presence and levels of autoantibodies was measured in serum samples from 50 patients with moderate to severe RA who had inadequate response to Methotrexate (average age 51.2, 86% of female, 78% of white race patients). Control cohort involved serum from 42 healthy age matched patients (average age 48.6, 50% of female, 60% of white race patients). Denaturation of the type II collagen was performed by heat treatment for 15 min at 72 °C. Improvement of assay sensitivity was investigated by measurements of autoimmunity levels against denatured and native type II collagen. Assay specificity was assessed by comparison of presence of autoantibodies against type II collagen versus albumin (non-sense control). The normality of data distribution was checked with Shapiro-Wilk test, significance between cohorts with Mann-Whitney nonparametric test and correlations with Spearman’s correlation coefficient (rho).Results:Serum levels of autoantibodies against denatured type II collagen were significantly higher in RA patients than in healthy controls (P < 0.0001) (Figure 1). We observed nearly 4-fold difference between both cohorts. Denaturation of type II collagen showed high improvement of assay sensitivity and increased accessibility of collagen for binding of autoantibodies. Developed assay showed specificity for detection of type II collagen autoantibodies by displaying no levels of autoimmunity against other control proteins (albumin). The levels of serum autoantibodies correlated significantly (P < 0.03) with patient disease activity (DAS28) at baseline, displaying rho = 0.3. Our assay showed good technical performance with acceptable inter- and intra- assay variations (18% and 5% respectively).Figure 1.Differences in serum levels of autoantibodies against denatured type II collagen between healthy controls and patients with Rheumatoid Arthritis.Conclusion:Present findings show that patients with RA carry upregulated levels of circulating autoantibodies directed against type II collagen. Heat-treatment of type II collagen increased exposure of immunogenic epitopes of collagen and enabled for more sensitive detection of autoantibodies directly in patient serum. Developed assay demonstrated potential for specific detection of autoantibodies and may provide additional diagnostic value in RA patients.Disclosure of Interests:Patryk Drobinski: None declared, Elijah Aighobahi: None declared, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S, Morten Karsdal Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S
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