Research paper
The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

https://doi.org/10.1016/j.jim.2017.11.004Get rights and content

Highlights

  • A peptide panel for detection of anti-citrullinated protein antibodies is developed

  • The peptide panel reacts with high sensitivity and high specificity

  • The peptide panel is able to compete with commercially available assay

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics).

A primary panel (peptides 814, 33062 and 33156) was identified, which obtained a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90%. These findings indicate that the generated peptide panel is optimal for ACPA detection and able to compete with commercial available assays. Collectively, this study may contribute to characterize autoimmunity towards citrullinated proteins and to the development of new and improved diagnostic assays for detection of ACPA and determination of RA.

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).

References (62)

  • E. Berglin et al.

    A combination of autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus antigens is strongly associated with future onset of rheumatoid arthritis

    Arthritis Res. Ther.

    (2004)
  • E. Berglin et al.

    Radiological outcome in rheumatoid arthritis is predicted by presence of antibodies against cyclic citrullinated peptide before and at disease onset, and by IgA-RF at disease onset

    Ann. Rheum. Dis.

    (2006)
  • N. Bizzaro et al.

    Analytical and diagnostic characteristics of 11 2nd- and 3rd-generation immunoenzymatic methods for the detection of antibodies to citrullinated proteins

    Clin. Chem.

    (2007)
  • M.A. van Boekel et al.

    Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

    Arthritis Res.

    (2002)
  • H. Burkhardt et al.

    Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis

    Eur. J. Immunol.

    (2005)
  • D. Coenen et al.

    Technical and diagnostic performance of 6 assays for the measurement of citrullinated protein/peptide antibodies in the diagnosis of rheumatoid arthritis

    Clin. Chem.

    (2007)
  • M.L. Correia et al.

    Comparison of three anti-CCP antibody tests and rheumatoid factor in RA and control patients

    Clin. Rev. Allergy Immunol.

    (2008)
  • C.E. Dam et al.

    The dependency on neighboring amino acids for reactivity of anti-citrullinated protein antibodies to citrullinated proteins

    Scand. J. Clin. Lab. Invest.

    (2016)
  • T. Dorner et al.

    Rheumatoid factor revisited

    Curr. Opin. Rheumatol.

    (2004)
  • O. Elkayam et al.

    Positive anti-cyclic citrullinated proteins and rheumatoid factor during active lung tuberculosis

    Ann. Rheum. Dis.

    (2006)
  • N. Fabien et al.

    Prevalence of autoantibodies to cyclic citrullinated peptide in patients with rheumatic diseases other than rheumatoid arthritis: a French multicenter study

    Clin. Rev. Allergy Immunol.

    (2008)
  • F.A. van Gaalen et al.

    A comparison of the diagnostic accuracy and prognostic value of the first and second anti-cyclic citrullinated peptides (CCP1 and CCP2) autoantibody tests for rheumatoid arthritis

    Ann. Rheum. Dis.

    (2005)
  • A.H. van der Helm-van Mil et al.

    Validation of a prediction rule for disease outcome in patients with recent-onset undifferentiated arthritis: moving toward individualized treatment decision-making

    Arthritis Rheum.

    (2008)
  • A. Ioan-Facsinay et al.

    Anti-cyclic citrullinated peptide antibodies are a collection of anti-citrullinated protein antibodies and contain overlapping and non-overlapping reactivities

    Ann. Rheum. Dis.

    (2011)
  • P. Kakumanu et al.

    Patients with pulmonary tuberculosis are frequently positive for anti-cyclic citrullinated peptide antibodies, but their sera also react with unmodified arginine-containing peptide

    Arthritis Rheum.

    (2008)
  • P. Kakumanu et al.

    Citrulline dependence of anti-cyclic citrullinated peptide antibodies in systemic lupus erythematosus as a marker of deforming/erosive arthritis

    J. Rheumatol.

    (2009)
  • A. Kinloch et al.

    Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis

    Arthritis Res. Ther.

    (2005)
  • T. Kogure et al.

    Insights to clinical use of serial determination in titers of cyclic citrullinated peptide autoantibodies

    Mediat. Inflamm.

    (2007)
  • M.C. Levesque et al.

    Anti-cyclic citrullinated peptide testing for the diagnosis of rheumatoid arthritis and the quest for improved sensitivity and predictive value

    Arthritis Rheum.

    (2009)
  • K.P. Machold et al.

    Very recent onset rheumatoid arthritis: clinical and serological patient characteristics associated with radiographic progression over the first years of disease

    Rheumatology (Oxford)

    (2007)
  • Y. Morita et al.

    Anti-cyclic citrullinated peptide antibody in systemic sclerosis

    Clin. Exp. Rheumatol.

    (2008)
  • Cited by (17)

    • Changes in future rheumatoid arthritis treatment in the light of Epstein-Barr virus infection

      2023, Translational Autoimmunity: Volume 6: Advances in Autoimmune Rheumatic Diseases
    • Preparation and characterization of cyclic citrullinated peptide-immobilized latex beads for measurement of anti-citrillinated protein antibody through latex particle-enhanced turbidimetric immunoassay

      2021, Journal of Chromatography A
      Citation Excerpt :

      Latterly, CCP, including 21 amino acid residues, is synthesized, in which amino acids 3 and 16 are cyclized by disulfide bonds, and this ring structure helps to expose the active center of citrulline adequately [19]. The sensitivity of the ACPA against CCP test is further increased with the development of new-style CCP [20]. Recently, ACPA against CCP test is commercially available test and used in the clinical diagnosis.

    • Label-free electrochemical impedimetric immunosensor for sensitive detection of IgM rheumatoid factor in human serum

      2019, Biosensors and Bioelectronics
      Citation Excerpt :

      Studies have recognized some of the causal pathways and target proteins that lead to AAb generation in RA (Sweet et al., 2013). The AAb commonly detected in the serum of RA patients consist of rheumatoid factors (RF) and anti-citrullinated peptide antibodies (ACPAs) (Trier et al., 2018; Aletaha et al., 2010). RF primarily consist of IgM, IgA, and IgG antibodies directed against the Fc fragment within the patient's own IgG antibody molecules (Aletaha et al., 2010; Westwood et al., 2006).

    • Neurodegeneration meets immunology – A chemical biology perspective

      2019, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      Thus, in this section, we will discuss the role of PTM in neurodegeneration and autoimmunity as well as chemical biology methods to investigate them. To date, there are several methods to investigate PTMs of proteins in complex samples/body fluids such as plasma/serum,106,107 synovial fluid,108,109 or CSF.110–113 Common techniques involve Western blotting with anti-PTM antibodies (in vitro),114 radioactive isotope labeling of target proteins (in vitro) and chemical derivatization with or without bioorthogonal groups/affinity-based proteomic profiling (in vitro and in vivo) coupled to tandem mass spectrometry (MS2).114–117

    • Reducing the risk of misdiagnosis of indirect ELISA by normalizing serum-specific background noise: The example of detecting anti-FGFR3 autoantibodies

      2019, Journal of Immunological Methods
      Citation Excerpt :

      Thus, we infer that the ELISA community has either not yet been reached or inadequately convinced. In a recent article, Haberland et al. (2018) nicely summarized the ongoing discussion and speculate whether calculating the difference between coated and non-coated wells (as already used in some studies (Trier et al., 2016, 2018a,b)) would resolve or increase the issue. They kindly invited the research community to contribute with ideas and wet lab experiments.

    • Management of Rheumatoid Arthritis and the Pharmacist's Role

      2019, Encyclopedia of Pharmacy Practice and Clinical Pharmacy: Volumes 1-3
    View all citing articles on Scopus
    View full text