High Sensitivity Detection of Anti-DFS70 Antibodies by Radioimmunoprecipitation Assay (RIPA).

BACKGROUND: Autoantibodies against the chromosome associated protein DFS70/LEDGF (dense fine speckled 70/ lens epithelial growth factor; anti-DSF70) are increasingly being regarded as biomarkers for the diagnostic exclusion of systemic autoimmune rheumatic diseases (SARD). In routine ANA screening by indirect immunofluores-cence (IIFT) tests the presence of anti-DFS70 may first be presumed because of their characteristic immunofluo-rescence pattern (AC-2 pattern) and then be confirmed by antigen specific assays, a sequential approach, which may underestimate the prevalence of anti-DFS70 because of the inherent shortcomings of the ANA-IIFT. We therefore, for the first time, determined the prevalence of anti-DFS70 in patient sera by means of a sensitive and specific radioimmunoprecipitation assay (RIPA) as compared to a commercial ELISA. METHODS: Blood specimens referred for routine ANA screening (n = 1.100, ANA-Series) or for basic clinical chemistry tests (n = 350, CC-Series) were assayed for the prevalence of anti-DFS70 by RIPA using 35S-methionine labelled full-length DFS70 (FL-DFS70) as well as a C-terminal DFS70 fragment (CT-DFS70) generated by in vitro transcription/translation (ivTT) of the respective cDNAs. ELISA was performed using an anti-DFS70 test-kit (Eu-roimmun) and ANA-IIFT by means of commercial HEp-2 cells (INOVA) and appropriately chessboard titrated conjugates (Dianova). Accessory SARD markers (anti-dsDNA, anti-ENA) were determined in sera positive for anti-DFS70. RESULTS: The detection of anti-DFS70 by RIPA was considerably more sensitive than by ELISA, resulting in an overall detection rate of 9.0% (ANA-Series) and 8.0% (CC-Series) compared to ELISA revealing 4.6% (ANA-Se-ries) and 2.6% (CC-Series) anti-DFS70 positive sera. Of 99 RIPA reactive sera (ANA-Series) 72% were reactive against anti-FL-DFS70, 93% against CT-DFS70, polyspecific antibodies coexisted in 65%, reacting with both antigen specificities, 28% showed monospecific reaction with CT-DFS70 and 7% monospecific with FL-DFS70, indicating also the possible existence of antibodies specific for N-terminal epitopes in DSF70. Similar frequencies were seen in sera of the CC-series. The RIPA measured antibody concentrations (Rratio) obtained with FL-DSF70 antigen and CT-DSF70 antigen showed a correlation. There was also a correlation between the IIFT-ANA titers and Rratio found by RIPA. The consensus of suspected AC-2 pattern in ANA-IIFT and anti-DFS70 measured by RIPA was about 80%. No significant correlation existed between the antibody concentrations measured by RIPA and ELISA. Additional SARD markers were present in 24% of anti-DFS70 positive sera referred for ANA screening. No additional markers were seen in sera of the CC-Series. CONCLUSIONS: RIPA constitutes a highly-sensitive assay for detection of anti-DFS70 in human sera. ANA-IIFT screening performed under consideration of the AC-2 pattern for verification of antibodies to DFS70 under routine conditions may incorrectly estimate a considerable number of not only low but also high titer anti-DSF70 positive sera. The significance of RIPA reactive antibodies, especially of low titer range, in the context of SARD and healthy individuals now has to be scrutinized in further clinical studies.

Autoantibodies Against DFS70/LEDGF Exclusion Markers for Systemic Autoimmune Rheumatic Diseases (SARD).

BACKGROUND: Antinuclear antibodies (ANA) are considered as a key serological feature of systemic autoimmune rheumatic diseases (SARD) which include syndromes like systemic lupus erythematodes (SLE), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), Sjögren's syndrome (SS) or dermatomyositis/polymyositis (DM/PM). ANA, commonly detected by indirect immunofluorescence assays on HEp-2 cells (IF-ANA), recommended as the screening test of choice (ACR), comprise a plethora of antibody specificities, a part of which are important serological markers of the diagnostic armamentarium in SARD. However, the applicability of IF-ANA as global screening test is hampered by its limited diagnostic specificity for resulting positive in up to 20% of apparently healthy individuals. About half of IF-ANA in healthy individuals target the chromosome associated protein DFS70/LEDGF, which tethers transcriptional coactivators or lentiviral integrases to transcriptionally active chromatin moieties and induces pro-survival stress factor transcriptions. Because of their rare prevalence in SARD patients, isolated anti-DFS70 antibodies are being increasingly considered as important biomarker to exclude the diagnosis of SARD. METHODS: Scrutinizing the relevant articles cited in NCBI concerning the DFS70/LEDGF protein, the diverse methods of anti-DFS70 determination in human sera supplemented by own experiences and critical review of the complete literature relevant to anti-DFS70 and SARD. RESULTS: Antibodies to DFS70/LEDGF (anti-DFS70), disclosed by IF-ANA, are characterized by a dense fine speckled (DFS) nucleoplasmic fluorescence pattern (DFS-ANA) accompanied by a striking staining of the condensed chromosomes in mitotic cells. By means of various methods anti-DFS70 may be found in 7.8 ± 6.2% (MD 7.6%) of apparently healthy individuals, may sometimes display rather high antibody titers and antibody carriers do not seem to manifest SARD symptoms within a five year interval. Their prevalence in non-selected cohorts originating from routine IF-ANA screenings (38643 tested individuals) fluctuates between 0.8 and 8.4% (MD 1.7%), depending on patient selection criteria and test performance. The proper appreciation of these data is hampered partially because of missing verification of antibody specificities partially by lack of specifications of associated disease or accompanying SARD specific marker antibodies. A metaanalysis of five studies including 1243 SARD patients confirms the rare mean prevalence of solitary anti-DFS70 (0.7 ± 0.9%, MD 0.45%) in SARD patients. The mean prevalence of anti-DFS70 accompanied by SARD specific markers is 3.8 ± 2.9% (MD 2.9%). In patients exclusively harbouring anti-DFS70 the likelihood ratio (LR+) for the absence of SARD approaches a significant value of 10.9. CONCLUSIONS: Since anti-DFS70 according to the available data may being regarded as a possible biomarker for ruling out the diagnosis of a systemic autoimmune rheumatic disease, it seems to be indispensable to identify properly DFS-ANA patterns in the routine IF-ANA screening, to confirm the anti-DFS70 specificity by appropriate confirmation assays and to communicate the results with annotating comments to the clinician, in order to ameliorate the proper assessment of the pathological significance of serological results and the selection of adequate follow-up investigations.

Autoantibodies against inosine-5'-monophosphate dehydrogenase 2--characteristics and prevalence in patients with HCV-infection.

BACKGROUND: Indirect immunofluorescence (IIFT) on in house HEp-2 cell preparations revealed a novel antibody giving a granular cytoplasmic pattern not described before, which on two commercial cell preparations revealed a "rings and rods" pattern. This pattern was also observed in four HCV-RNA carriers and prompted the identification of the reactive antigen and the evaluation of the antibody prevalence in HCV-RNA carriers and control groups. METHODS: The antigen's molecular weight was determined by radioimmunoprecipitation of 35S-methionine labeled cell proteins. Expression library screening and sequencing was performed by standard techniques using an oligo(dT)-primed human HeLa cell cDNA expression library. Antibodies against the novel antigen Inositol-5'-monophosphatdehydrogenase 2 (IMPDH2) were analyzed by IIFT, western blot, line blot, and radioimmunoprecipitation assay (RIPA). IIFT was performed on commercial HEp-2 cells and cells cultivated in house for 24 - 60 hours, with or without the IMPDH2 inhibitors mycophenolic acid (MPA) or ribavirin, and subjected to various fixation conditions. Western and line blots were performed with IMPDH2 synthesized in E. coli, RIPA with 35S-methionine-IMPDH2 from in vitro transcription/translation products. Sera screened were positive for HCV-RNA (108), HBV-DNA (100), anti-mitochondrial (31), anti-actin (42), and anti-nuclear antibodies (51) and negative for HCV-RNA (100) and blood donors (100). RESULTS: IMPDH2 is capable of considerable intracellular rearrangements (upon action of inhibitors like MPA and ribavirin), which explains the contrasting immunofluorescence patterns in cells from different sources. By RIPA, proven to be the sole assay suitable for screening of anti-IMPDH2 in human sera, autoantibodies were found in 35.2% of HCV-RNA carriers and in low concentrations in 31% of anti-actin positive patients suspicious of autoimmune hepatitis. Antibodies reacted preferentially with conformational epitopes. Compared to the low concentration of anti-IMPDH2 found in other disease groups, high antibody concentrations were observed in HCV-RNA carriers. CONCLUSIONS: The common occurrence of anti-IMPDH2 in HCV-RNA carriers may be related to ribavirin therapy, causing intracellular aggregation of IMPDH2 thereby altering its immunogenicity. In this study the "rods and rings" immunofluorescence pattern observed could be ascribed to anti-IMPDH2. Anti-IMPDH2 may cause difficulties in interpretation of immunofluorescence patterns in routine autoantibody testing.