Immunoblotting detection of so-called 'antikeratin antibodies': a new assay for the diagnosis of rheumatoid arthritis.

Gomès-Daudrix, V; Sebbag, Mireille; Girbal, E; Vincent, Christian; Simon, Michel; Rakotoarivony, J; Abbal, M.; Fournie, B.; Serre, Guy

doi:10.1136/ard.53.11.735

Cited by 25 publications

(14 citation statements)

References 33 publications

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“…Previously, immunoblotting has been used to measure so-called 'AKA' using epidermal filaggrin [6] and extracts of rat esophagus as antigens [23]. Immunoblotting is, however, not suited to the study of large numbers of patients and controls; moreover, it is at best only semiquantitative as in- Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Palosuo

Lukka

Alenius

et al. 1998

Int Arch Allergy Immunol

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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.

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Section: Discussionmentioning

confidence: 99%

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

Palosuo

Lukka

Alenius

et al. 1998

Int Arch Allergy Immunol

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“…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).…”

Section: R Heumatoid Arthritis (Ra)mentioning

confidence: 99%

“…Human epidermis extracts enriched in neutral/acidic filaggrin and rat esophagus epithelium extracts containing the AFA-targeted antigenic proteins were separated by SDS-PAGE and nondenaturing PAGE, respectively, using a PhastSystem (Pharmacia Biotech, Uppsala, Sweden) as previously described (3,5). Proteins were then electrotransferred onto reinforced nitrocellulose membranes (Hybond-C extra; Amersham, Little Chalfont, U.K.).…”

Section: Immunoblottingmentioning

confidence: 99%

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

Masson-Bessière

Sebbag

Girbal-Neuhauser

et al. 2001

The Journal of Immunology

583

502

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IgG antifilaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis. In epithelial tissues, they recognize citrulline-bearing epitopes present on various molecular forms of (pro)filaggrin. Histological analysis of rheumatoid synovial membranes with an Ab to citrulline showed labeling of interstitial amorphous deposits and mononuclear cells of various types. Immunochemical analysis of exhaustive sequential extracts of the same tissues showed that they contain several deiminated (citrulline containing) proteins. Among them, two proteins, p64–78 and p55–61, present in urea-DTT and guanidine extracts, were shown by immunoblotting to be specifically targeted by AFA. By amino-terminal sequencing the proteins were identified as deiminated forms of the α- and β-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the Aα- and/or to the Bβ-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the Aα- and Bβ-chains of human fibrinogen only after deimination of the molecules by a peptidylarginine deiminase. Autoantibodies affinity purified from a pool of RA sera onto deiminated fibrinogen were reactive toward all of the epithelial and synovial targets of AFA. This confirmed that the autoantibodies to the deiminated Aα-and Bβ-chains of fibrinogen, the autoantibodies to the synovial proteins p64–78 and p55–61, and, lastly, AFA, constitute largely overlapping autoantibody populations. These results show that deiminated forms of fibrin deposited in the rheumatoid synovial membranes are the major target of AFA. They suggest that autoimmunization against deiminated fibrin is a critical step in RA pathogenesis.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Chapuy‐Regaud

Nogueira

Clavel

et al. 2005

Clinical and Experimental Immunology

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SummaryIn the rheumatoid synovium, deiminated ('citrullinated') forms of fibrin are the major targets of IgG autoantibodies to citrullinated proteins (ACPA), the most specific serological markers of rheumatoid arthritis (RA). To further the characterization of ACPA, we determined their subclass distribution. From a previously validated highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) onto in vitro deiminated human fibrinogen ----antihuman fibrin(ogen) autoantibodies (AhFibA)-ELISA ----we derived and calibrated four ELISAs, using monoclonal antibodies to each of the four IgG subclasses, to determine the proportions of AhFibA subclasses in the sera. A series of 186 serum samples from RA patients was analysed. All AhFibA-positive sera contained IgG1-AhFibA, which reached the highest titres and accounted for more than 80% of AhFibA in three-quarters of the sera. One or two other subclasses were associated with IgG1 in 39% of the sera, IgG4-AhFibA being observed much more frequently and at higher titres than IgG3-or IgG2-AhFibA. IgG1 alone or IgG(1 + 4)-AhFibA were the AhFibA subclass profiles found in more than 80% of patients. AhFibA are mainly IgG1 and, to a lesser extent, IgG4. Such IgG subclass profiles may influence the effector phases of the immunological conflict between ACPA and deiminated fibrin that takes place specifically in the rheumatoid synovium and therefore may play a critical role in the self-maintenance of rheumatoid inflammation.

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Immunoblotting detection of so-called 'antikeratin antibodies': a new assay for the diagnosis of rheumatoid arthritis.

Cited by 25 publications

References 33 publications

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

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

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

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

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