Purification of filaggrin from human epidermis and measurement of antifilaggrin autoantibodies in sera from patients with rheumatoid arthritis by an enzyme-linked immunosorbent assay

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 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.