Research Article: Development of a multiplex assay based on chimeric citrullinated peptides as proof of concept for diagnosis of rheumatoid arthritis

Date Published: May 2, 2019

Publisher: Public Library of Science

Author(s): Cristina García-Moreno, María José Gómara, María José Bleda, Raimon Sanmartí, Isabel Haro, Salvatore V. Pizzo.


Anti-citrullinated peptide/protein antibodies (ACPAs) are the most specific serological biomarkers for rheumatoid arthritis (RA). They have both diagnostic and prognostic value, and are related to more aggressive joint disease in RA. However, a single biomarker cannot differentiate RA subtypes. So, simultaneous analysis of target citrullinated peptides, using a multiplex array based on chimeric peptides composed of several domains of human proteins, could be useful. In this work, eight chimeric peptides and the corresponding native arginine-containing control peptides were obtained by solid-phase peptide synthesis. The study included RA and psoriatic arthritis (PsA) patients attending the Rheumatology Unit of the Hospital Clinic in Barcelona, as well as healthy blood donors (BD) at the same hospital. Our main aim was to explore the diagnostic value of the novel multiplex array compared to a commercial ELISA-based ACPA assay in a serum-saving way. Using the combination of the eight chimeric peptide antigens in the multiplex array, 61.4% of the RA cohort were positive for 3 or more peptides; while, the healthy BD and PsA cohorts did not show any reactivity with the tested peptides. These results indicate that we have developed a highly specific multiplex assay based of chimeric citrullinated peptides derived from filaggrin, fibrin, vimentin and human enolase proteins for the detection of ACPAs in a serum-saving way.

Partial Text

Rheumatoid arthritis (RA) is a chronic and incapacitating inflammatory disease of the joints which is estimated to affect 0.5%-1% of the population worldwide. Long-lasting and the more severe cases can also develop into a systemic disease and have extra-articular effects [1,2]. As symptoms do not always appear in the early stages of the disease, there is a clear need to improve both the precision of specific tests for its diagnosis, and its early differentiation from other rheumatic diseases that affect the articulations and connective tissue. This is especially so in the case of patients with a poor prognosis or those in the early developmental stages of the disease.

The work was approved by the Ethics Committees of the Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, and of the Hospital Clinic, Barcelona, Spain. All methods were performed in accordance with the relevant IQAC-CSIC guidelines and regulations. Written consent was obtained from all participants and suitably informed.

As previously reported [6,7], the identification of novel citrullinated antigens that could supplement or complement ACPA-based existing tests, is required. A challenging approach consists of analyzing ACPA profiles using multiple citrullinated peptide antigens simultaneously. In the present work, considering the increased antigenicity previously observed with multimeric peptides bearing different epitope peptide sequences within the same molecule, we designed and synthesized three new chimeric peptides in the solid phase. They were composed of citrullinated peptides derived from filaggrin (cyclic filaggrin peptide, constituting the CCP1 test), fibrin (617–631), vimentin (47–72) and enolase (CEP-1), and herein we refer to them as chimeric fibrin/enolase citrullinated peptide (CFECP), chimeric vimentin/enolase citrullinated peptide (CVECP) and chimeric enolase/filaggrin citrullinated peptide (CEFCP). The analytical characterization of these three novel chimeric peptides using UPLC-MS is shown in the Supplementary material (Figs A, B and C in S1 File) and the primary structure of all the chimeric citrullinated peptides studied in the present work is shown in Fig 1. Firstly, a comparative ELISA assay was performed in cohorts of 100 RA and 87 BD sera using CFECP, CVECP and CEFCP as coating antigens; as was previously performed with the chimeric peptides CFFCP1, CFFCP2, CFFCP3, CFVCP and CVFCP [6, 7]. The values of the area under the ROC curve (RA vs BD) are shown in Fig 2: CEFCP: 0.78; CVECP: 0.71; CFECP: 0.78. Using cut-off values that yield a specificity of 98.85% with respect to BD, the sensitivity of the ELISAs was 63% for CEFCP, 37% for CVECP, and 56% for CFECP; with PPV > 97.4% in all three tests and NPV between 57.7% and 69.9%.

Here, we have develop a novel multiplex platform composed of eight chimeric citrullinated peptides derived from human proteins that abound in rheumatoid synovial, and we have evaluated its suitability to detect anti-citrullinated peptide/protein antibodies (ACPAs) in serum samples of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients, as well as in healthy blood donors. Our results demonstrate that the platform has a high specificity for ACPA detection, especially taking into account that the control group consisted of patients affected by PsA, an inflammatory disease whose clinical presentation can simulate that of RA. When comparing with the commercial ELISA-based test (CCP3), it is true that ACPA reactivity was only detected by the multiplex array in one CCP3-negative RA serum sample. However, this in-house multiplex system allowed us to control for background reactivity to arginine and worked in a serum-saving way, compared to CCP3. In conclusion, a highly citrulline-specific multiplex assay based on chimeric citrullinated peptides derived from filaggrin, fibrin, vimentin and human enolase proteins for the detection of ACPAs has been developed herein. In order to improve sensitivity and expand future use in RA diagnosis, peptides bearing other non-proteinogenic amino acids could be designed, synthesized and incorporated into the platform as novel antigenic substrates for further development of the multiplex system.




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