Research paperThe use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis
Introduction
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease that affects approximately 1% of the population, leading to significant morbidity and increased mortality (Scott et al., 2010, Sokka et al., 2008). A characteristic feature of RA is the presence of rheumatoid factors (RFs) and the more disease-specific autoantibodies anti-citrullinated peptide antibodies (ACPAs) (Song and Kang, 2010). While IgA/IgM RFs are directed against the Fc portion of IgGs, ACPAs are directed against citrullinated targets (Dam et al., 2016, Kinloch et al., 2005, Song and Kang, 2010). Citrullination is a posttranslational modification catalyzed by the enzyme peptidyl arginine deiminase, where the positively charged amino acid arginine is modified into the neutral amino acid citrulline (Cit) (Tarcsa et al., 1996).
The diagnosis of RA depends primarily on clinical manifestations of the disease supported by the detection of the serological biomarkers RF and ACPA (Aletaha et al., 2010, van Boekel et al., 2002). Originally, the only serological test routinely used was the determination of RFs. The sensitivity of RF for diagnosis is 50–90% and specificity is 50–95%, dependent on the method applied and differences in population tested (Dorner et al., 2004, Nishimura et al., 2007, Shmerling and Delbanco, 1991). RFs may also be detected in individuals affected by infections, other autoimmune diseases, e.g. such as systemic lupus erythematosus (SLE), mixed connective tissue disease, Sjögren's syndrome (SjS), and occasionally in healthy individuals. Thus, more RA-specific serological markers are warranted (Nell et al., 2005).
ACPAs and the dependency of Cit for antibody reactivity were originally described by Schellekens and colleagues (Schellekens et al., 1998). However, the role of ACPAs and their respective citrullinated epitopes in the pathogenesis of RA still remain unclear. ACPAs are associated with an increased risk of developing RA and have been reported to be present before the onset of RA symptoms (Berglin et al., 2004, Nielen et al., 2004, Rantapaa-Dahlqvist et al., 2003). Moreover, the presence of ACPAs have been suggested to be associated with a more severe disease course, as ACPAs are associated with structural damage of tissues, radiographic progression and poorer response to therapy (Berglin et al., 2006, del Val del et al., 2006, Machold et al., 2007, Quinn et al., 2006).
Citrulline-dependent antibodies were initially described by Nienhuis and colleagues and were termed anti-perinuclear factors (Nienhuis and Mandema, 1964). Later Sebbag and colleagues demonstrated that these antibodies belonged to a family of autoantibodies directed against citrullinated filaggrin (Sebbag et al., 1995). While filaggrin is not present in the synovium (Baeten et al., 2001), several citrullinated proteins are present here, including, but not restricted to fibrinogen and fibronectin. In fact, multiple citrullinated protein targets have been identified in RA, e.g. vimentin, fibrin, α-enolase and collagen (Burkhardt et al., 2005, Kinloch et al., 2005, Schellekens et al., 1998, Sebbag et al., 2006, Vossenaar et al., 2004).
For clinical detection of ACPAs, most testing is performed using commercially available assays that utilize synthetic cyclic citrullinated peptides (CCP) as antigens, although for proprietary reasons, the specific antigens present within these assays are unknown. The first peptide-based enzyme-linked immunosorbent assay (ELISA) test for detection of ACPA was based on a synthetic linear citrullinated peptide derived from human filaggrin. To improve antibody recognition, a cyclic version of the peptide was employed, which showed higher sensitivities and specificities compared to the linear peptide version (Schellekens et al., 2000). The assay detected CCP antibodies in 53% of established RA patients with a 96% specificity. Following this, peptide libraries were screened for better epitopes, leading to the 2nd (CCP2) and 3rd generation CCP (CCP3) of assays, and in theory development of assays detecting different classes of ACPAs (Aggarwal et al., 2009, Saraux et al., 2003, Vander Cruyssen et al., 2008, Vittecoq et al., 2004). Apart from the main difference in peptide substrate, both CCP2 and CCP3 use similar screening approaches. Most studies, however, show no evident improvement of CCP3 compared to CCP2 assays (Aggarwal et al., 2009, Coenen et al., 2007, Correia et al., 2008, Santiago et al., 2008) as similar sensitivities (68–79%) and specificities (86–96%) are obtained (Bizzaro et al., 2007, Kogure et al., 2007, Lutteri et al., 2007, Nishimura et al., 2007, Santiago et al., 2008, van Gaalen et al., 2005). Whereas 1st, 2nd and 3rd generation of CCP assays only detect ACPA IgGs, does the CCP3.1 assay marketed by Inova Diagnostics detect both IgG and IgA ACPAs in an effort to increase assay sensitivity (Coenen et al., 2007). However, currently CCP2 assays are still most commonly used in the clinical and research assays.
Despite the high specificity of ACPAs for RA, individuals with other rheumatic diseases, including psoriatic arthritis, SLE, SjS, systemic sclerosis and primary biliary cirrhosis, may be positive for ACPA, although the types of ACPA testing used in these studies have varied widely (Kakumanu et al., 2009, Morita et al., 2008, Payet et al., 2014, Santiago et al., 2008). Finally, ACPAs have been detected in 30% of individuals with active pulmonary tuberculosis and no evidence of articular RA (Elkayam et al., 2006, Kakumanu et al., 2008). However, further studies revealed that in these patients with tuberculosis, ACPA reactivity may have been due to non-citrulline-specific antibody reactivity, as these individuals have similar antibody reactivity levels to the non-citrullinated arginine residues (Elkayam et al., 2006, Kakumanu et al., 2008).
In this study, we analyzed RA reactivity to citrullinated peptides in order to identify substrates for ACPA detection. Moreover, these findings were compared to the commercially available assays QUANTA Lite ® CCP3.1 IgA/IgG (Inova Diagnostics) (3rd generation) and CCPlus ® (EuroDiagnostica) (2nd generation assay).
Section snippets
Patient sera
Sera from 126 RA patients diagnosed according to the American College of Rheumatology (ACR) classification criteria were enrolled in this study (Aletaha et al., 2010, Arnett et al., 1988). These sera were from the Department of Rheumatology, Glostrup Hospital, Department of Rheumatology, Frederiksberg Hospital, Department of Rheumatology, Odense University Hospital and Epidemiology, Biostatistics and Bio-demography, Institute of Public Health, University of Southern Denmark. In addition, 57
Screening of citrullinated proteoglycan peptides
To identify citrullinated targets as substrates for ACPA detection, citrullinated proteoglycan peptides were screened for reactivity. For preliminary screenings, the reactivity of 10 anti-CCP2-positive and 10 healthy control sera to the citrullinated proteoglycan peptides was analyzed by modified ELISA using resin-bound peptides.
As seen (Fig. 1), the anti-CCP2-positive sera reacted with 23 out of 24 peptides, only peptide 33056 was not recognized, although significant antibody reactivity only
Discussion
ACPAs are important serological markers in the diagnosis of RA (van der Helm-van Mil et al., 2008). Despite intense efforts to standardize ACPA detection (Rottmann, 2010), significant differences exist between ACPA assays Bizzaro et al., 2007). While the first generation CCP assay was based on a single peptide (Schellekens et al., 2000), the following generations of assays were identified by screening complex peptide libraries and combinatorial peptide engineering (Levesque et al., 2009).
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