Identification of citrullinated rheumatoid arthritis‐specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay

Abstract

Objective. To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format.Methods. Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods.Results. Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P ‫؍‬ 0.0047).Conclusion. Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

Deiminated Epstein‐Barr virus nuclear antigen 1 is a target of anti–citrullinated protein antibodies in rheumatoid arthritis

“…Anti-CCP antibodies were detected with a commercial kit (QUANTA Lite CCP, Inova Diagnostics, San Diego, CA), according to the manufacturer's instructions. Antifilaggrin-derived deiminated peptide antibodies were detected using a research version of a line immunoassay (INNO-LIA RA; Innogenetics, Alpharetta, GA) based on 2 citrullinated synthetic peptides derived from human filaggrin (26). Protein G-purified immunoglobulins from normal human sera were included as negative controls.…”

Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis

“…In addition, two synthetic cFil-derived peptides as well their unmodified isoforms were provided and used at 2.5 g/ml. These peptides were selected on the basis of recognition by AFA (46) and had the following sequences (X ϭ citrulline, i.e., deiminated arginine): HSAS QDGQDTIXGHPGSS (filpep 1) and DSGHXGYSGSQASQDNEGH (filpep 2).…”

Cross‐reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

“…4,10 Antibodies specific for citrullinated proteins recognize a variety of citrullinated autoantigens, including a-enolase, fibrinogen, type II collagen, and vimentin, and are collectively identified as anticitrullinated peptide/protein antibodies (ACPA). [11][12][13][14][15] These ACPA responses, primarily characterized by IgG 1 and IgG 4 subtypes (99 and 98% of ACPA-positive RA patient sera, respectively), have been found to be present up to years before disease onset and to be highly specific (98%) and sensitive (up to 80%) for RA. [16][17][18][19][20][21] Thus, APCA are used as a supplement to clinical diagnosis of RA.…”

“…Especially the length of the antigenic binding region has been found to be essential for antibody reactivity. 9 In addition, amino acids surrounding citrulline have been shown to be essential for ACPA reactivity, and especially antigen binding regions containing citrulline residues flanked by neutral and relatively flexible structures, such as glycine and serine, 9,15,25,29,30 often show increased reactivity compared to peptides containing strongly charged residues or residues of fixed structure such as proline, as these contribute to a rigid structural conformation. In addition, structural homology of the amino acid residues surrounding citrulline has been described to be essential as well.…”