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.

Severe Myasthenic Manifestation of Leptospirosis Associated with New Sequence Type of Leptospira interrogans.

We report the rapid development of a myasthenic crisis as the first-time manifestation of myasthenia gravis. The symptoms developed in the course of acute leptospirosis associated with a new sequence type of Leptospira interrogans. Antibiotic treatment led to rapid amelioration of myasthenia.

Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients. METHODS AND FINDINGS: Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive). CONCLUSIONS: In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of anti-Sm antibodies.

Anti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen.

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3-10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11-16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-alpha) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-alpha peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-alpha and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-alpha peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-alpha peptide antibodies. These data indicate that anti-PM1-alpha antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-alpha ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.

Development of a CENP-A/CENP-B-specific immune response in a patient with systemic sclerosis.

Antibodies directed against an epitope motif on CENP-A have been shown to cross-react with mimotopes on other autoantigens and on Epstein-Barr nuclear antigen 1 (EBNA-1), suggesting a molecular mimicry. We describe here the gradual development of an anticentromere immune response in a patient with systemic sclerosis, which started from an antihistone response and was not mediated by molecular mimicry. Via an epitope on histone H3, the antibody response spread to a homologous epitope in the H3 homology domain of CENP-A. This was followed by an intramolecular epitope spreading to N-terminal peptides of CENP-A containing the known epitope motif G-P-X(1)-R-X(2). From there it spread to corresponding epitopes on CENP-B and to mimotopes of the major CENP-A epitope motif on other autoantigens including EBNA-1. Whether the D-penicillamine treatment received by this patient was involved in the triggering of this cascade remains a matter of speculation.