Objective Anti-double-stranded (ds) DNA and anti-C1q autoantibodies are useful tools in the assessment of disease activity and nephritis in systemic lupus erythematosus (SLE) patients. This study aimed to explore the utility of these antibodies along with anti-Ku antibodies in an oligoparametric model approach for the assessment of disease activity and lupus nephritis. Methods Samples from 261 well-characterized SLE patients were tested using chemiluminescent immunoassays (CIA) for anti-dsDNA and anti-Ku antibodies as well as by anti-C1q antibody ELISA (Inova Diagnostics, USA). Of these SLE patients, 26.4% had lupus nephritis (LN) at the time of blood draw or had a history of LN, and modified SLE disease activity index-2K (SLEDAI) scores were used to assess disease activity. Results All three antibodies demonstrated higher prevalence and higher antibody levels in active versus inactive SLE patients and in LN versus non-LN patients. When oligoparametric analysis was performed, the likelihood of LN and patients with active disease increased with dual and triple positivity. Conclusions Anti-dsDNA and anti-C1q antibodies are useful tools to identify disease activity and/or renal involvement in SLE patients. In addition, the combination of those antibodies in a two-parametric score might improve the clinical utility of those markers.

Objective We sought to evaluate different anti-double-stranded DNA assays for their performance characteristics in monitoring disease activity fluctuations in systemic lupus erythematosus (SLE). Methods 36 active SLE patients were followed monthly. At each study visit (total n = 371), blood was collected and disease activity was scored using the SELENA-SLEDAI (excluding anti-dsDNA or complement components) and by a physician's global assessment (PGA). Four anti-dsDNA tests were compared. Linear mixed-effects models with random intercept and fixed slopes were used to evaluate the relationship between the longitudinal fluctuations of disease activity and anti-dsDNA titers. Results At enrollment, positivity for QUANTA Lite and high-avidity anti-dsDNA assay was both 64% and significantly lower than anti-dsDNA positivity by QUANTA Flash (83%) and CLIFT (96%). Linear mixed-effects modeling indicated that the change in clinical SELENA-SLEDAI scores was associated with the titers of all anti-dsDNA with QUANTA Flash yielding the highest marginal R2 (0.15; p < 0.01). QUANTA Flash was the only anti-dsDNA assay significantly associated with the change in PGA (marginal R2 = 0.05; p < 0.01). Conclusion These data indicate that anti-dsDNA antibodies determined by QUANTA Flash have a value in monitoring SLE disease activity.

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Background:Protein-arginine deiminase (PAD) 4 enzymes play a central role in the pathogenesis of rheumatoid arthritis (RA) and represents an antigenic target. Among the five known family members (PAD1, PAD2, PAD3, PAD4 and PAD6), only PAD2, PAD3 and PAD4 have been described to have autoantigenic properties. Furtheremore, very little is known on the the isotype usage of these autoantibodies. Understanding the molecular basis of the anti-PAD antibody reponse has the potential to open novel approaches for precision medicine in RA.Objectives:The objectives of this study were to screen for the presence of antibodies to the five PAD family members and to evaluate the isotype usage of the anti-PAD4 response in RA.Methods:First, we developed a panel for the detection of anti-PAD IgG based on a particle-based multi-analyte technology (PMAT), that utilized paramagnetic particles coupled with the different human recombinant PAD proteins (PAD1, PAD2, PAD3, PAD4 and PAD6) and anti-human IgG conjugate. This panel was used to test sera from RA patients (n=33) and non-RA controls (n=36). The controls were comprised of apparently healthy individuals (n=10), and patients with infectious diseases (n=10), systemic lupus erythematosus (n=7), systemic sclerosis (n=9) and Sjogren’s syndrome (n=1). Next, the PAD4-coupled beads were tested with anti-human IgM, IgA and IgG conjugates on an extended cohort of RA patients (n=62) and the same non-RA controls.Results:All five anti-PAD IgG (Figure 1) demonstrated the ability to discriminate between RA patients and controls. At greater than 90% specificity, anti-PAD4 IgG, followed by anti-PAD3 IgG, showed the best diagnostic performance. Significantly higher levels of the five antibodies were observed in RAvs.controls (p-values of 0.0041, <0.0001, 0.0014, 0.0039, and 0.0140 for anti-PAD1, 2, 3, 4 and 6, respectively). Significant correlation was observed between all the antibodies, with the highest between anti-PAD1 and anti-PAD4 (Spearman´srho=0.87,p<0.0001) and the lowest between anti-PAD4 and anti-PAD2 (Spearman’srho=0.38,p=0.0015) and anti-PAD4 and anti-PAD6 (Spearman’srho=0.38,p=0.0011). While principal component analysis (PCA) (Figure 2) showed an association between all anti-PAD antibodies, there was further discrimination that displayed closer association between anti-PAD1, 3 and 4 on one hand, and between anti-PAD2 and 6. For the extended testing of anti-PAD4 with IgG, IgA and IgM, all three isotypes were identified in the sera of RA patients. Higher levels of the three isotypes were observed in RA patients with erosive disease when compared with the patients without erosion, but this association was only significant for anti-PAD4 IgA (p=0.0086).Figure 1.Receiver operating characteristics (ROC) analysis of the discrimination between rheumatoid arthritis (RA) and controls of IgG to protein-arginine deiminase (PAD) 1, PAD2, PAD3, PAD4 and PAD6. The area under the curve (AUC) values are shown in brackets for each biomarker.Abbreviations:TPF: true positive fraction; FPF: false positive fractionFigure 2.Two dimensional principal component analysis (PCA) plot of the anti-PAD levels in RA patients (n=33) and controls (n=36). Anti-PAD1, 3 and 4 have the main contribution to PC1, which explains 51.7% of the variance, and anti-PAD2 and 6 to PC2, that represents 20.8% of it.Abbreviations:PC: principal componentConclusion:Our study is the first to describe PAD1 and PAD6 as novel antigenic targets in RA and to demostrate that the anti-PAD4 B-cell immune response uses all three isotypes (IgG, IgA and IgM). The strong and significant association between anti-PAD4 IgA and joint erosion is of particular clinical relevance.Disclosure of Interests:Laura Martinez-Prat Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Mary Ann Aure Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Chelsea Bentow Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., David Lucia Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Marcos Lopez-Hoyos Consultant of: Inova Diagnostics, an in vitro diagnostics company., Michael Mahler Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company.

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