Purification of Filaggrin from Human Epidermis and Measurement of Antifilaggrin Autoantibodies in Sera from Patients with Rheumatoid Arthritis by an Enzyme-Linked Immunosorbent Assay

Abstract

Background: The so-called antikeratin antibody (AKA) and the antiperinuclear factor (APF) that recognize proteins related to human epidermal filaggrin belong to the most specific serological markers of rheumatoid arthritis (RA). However, assays for the detection of AKA and APF are currently based on immunofluorescence, a method that is subject to arbitrary interpretation and inadequate standardization of the substrates. Methods: Proteins extracted from human epidermis were separated by reversed-phase high-performance liquid chromatography (HPLC). Filaggrin-containing fractions, identified in immunoblotting by monoclonal antifilaggrin antibodies, were then subjected to gel filtration HPLC and, finally, to a second reversed-phase HPLC step. Tryptic digestion, amino acid sequencing and mass spectrometry were used to cornfirm the identity of the purified protein. Filaggrin was used as antigen in enzyme-linked immunosorbent assay (ELISA) to measure IgG class antifilaggrin antibodies. Results: The filaggrin preparation obtained gave a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, binding monoclonal antifilaggrin antibody in immunoblotting. Amino acid sequences of all 10 tryptic peptides analyzed were shown to originate from human filaggrin. Antifilaggrin antibody levels exceeded the 99th percentile level of 100 middle-aged blood donors in 26/55 (47%) RA sera. At a similar cutoff level 28/55 (51%) of the RA sera were positive in the AKA test. Of the 26 antifilaggrin-positive sera, 21 were also AKA-positive. Conclusion: Human filaggrin can be purified by standard biochemical techniques, despite the heterogeneity of the protein, and used in ELISA for testing autoantibodies to filaggrin. The sensitivity of the assay equals that of the AKA test.

The Major Synovial Targets of the Rheumatoid Arthritis-Specific Antifilaggrin Autoantibodies Are Deiminated Forms of the α- and β-Chains of Fibrin

“…Reflecting the humoral autoimmune processes occurring during RA is the presence of autoantibodies of diverse specificities in the serum of patients. Among these, antifilaggrin autoantibodies (AFA) are the most disease specific and are increasingly recognized as a useful diagnosis tool (1)(2)(3)(4)(5). AFA were originally described as IgG labeling the cornified layer of rat esophagus epithelium by indirect immunofluorescence and formerly called "anti-keratin Abs" (AKA) (6).…”

IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin

“…Several diagnostic immunoassays, based on the recognition of human or rat filaggrin or of citrulline-bearing peptides derived from human filaggrin, were developed [8][9][10][11][12][13][14] that, together with the classical 'AKA' and 'APF' indirect immunofluorescence tests, have allowed the collection of bioclinical data supporting the hypothesis of a critical role of ACPA in RA. In addition to having a high diagnostic specificity these autoantibodies appear early [15][16][17], even before the clinical onset of the disease [18][19][20], and are associated with the most severe forms of RA [21][22][23][24].…”

Clinical significance of anti-filaggrin antibody recognizing uncitrullinated filaggrin in rheumatoid arthritis

“…AFA is the autoantibody to filaggrin in human epidermis and detected in the RA patients by immunoblot analysis and ELISA using human epidermal extracts or synthetic citrullinated peptide as an antigen (Palosuo et al, 1998;Vincent et al, 1998;Schellekens et al, 2000). Although the specificity of the tests was high (92.0-95.4%), the sensitivity of the tests was variable (12.0-67.9%) probably due to the difference in source of filaggrin extracted or in sequences of citrullinated peptide from various providers.…”

“…Immunoblot tests using the filaggrin extract from human epidermis showed variable sensitivity ranging from 12.0-67.9%, with the specificity of 92.0-95.4% (Vincent et al, 1998;Slack et al, 1998). ELISA using filaggrin purified from human epidermis detected IgG AFA in 47% of RA patients (Palosuo et al, 1998). Recently, ELISA using a cyclic citrullinated synthetic peptide derived from the sequence of human filaggrin showed a higher specificity than other methods (Schellekens et al, 2000;Girelli et al, 2004).…”