Is alpha-actinin a target for pathogenic anti-DNA antibodies in lupus nephritis?

OBJECTIVE: Following recent reports that pathogenic murine anti-DNA antibodies bind to alpha-actinin, it was obviously of interest to assess the ability of human pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies to bind this antigen. Both human monoclonal anti-DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated. METHODS: An enzyme-linked immunosorbent assay was established to measure immunoglobulin binding to alpha-actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high-affinity human anti-dsDNA IgG hybridomas (RH14, B3, and DIL-6) and 7 human IgM anti-DNA hybridomas was also investigated. RESULTS: A greater proportion of anti-dsDNA IgG-binding antibodies purified from patients with renal disease bound to alpha-actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100-kd alpha-actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to alpha-actinin, while nonpathogenic DIL-6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to alpha-actinin. CONCLUSION: The results of our study support the findings of previous studies using murine anti-DNA monoclonal antibodies, which suggest that pathogenic anti-dsDNA antibodies cross-react with alpha-actinin.     

Association of α‐actinin–binding anti–double‐stranded DNA antibodies with lupus nephritis

Objective

Anti–double‐stranded DNA (anti‐dsDNA) antibodies may contribute to the pathogenesis of glomerulonephritis (GN) by cross‐reacting with α‐actinin in murine models and in some patients with systemic lupus erythematosus (SLE). We therefore sought to determine possible disease associations with serologic and clinical features and to characterize this new autoantibody specificity.

Methods

One hundred patients with SLE were recruited into this multicenter study, as well as 100 rheumatic disease controls and 2,100 healthy blood donors. Clinical disease was evaluated by the SLE Disease Activity Index (SLEDAI; excluding the anti‐DNA component). Anti‐dsDNA antibodies were detected by conventional enzyme‐linked immunosorbent assay (ELISA) and by a commercial enzyme immunoassay (EIA). Anti–α‐actinin antibodies were detected by ELISA, and their specificity was confirmed by Western blotting and by indirect immunofluorescence using rat kidney sections and mesangial cells as substrates. Highly positive sera were selected for absorption experiments and were affinity‐purified for cross‐reactivity studies and measurement of antibody avidity.

Results

Sera from 62 of the SLE patients had anti‐dsDNA antibodies; 21 of these sera also had anti–α‐actinin antibodies, as compared with 1 of the 38 sera without anti‐dsDNA antibodies. Of the 22 patients with anti–α‐actinin antibodies, 10 had GN, as compared with 14 of the 78 without anti–α‐actinin antibodies (P < 0.01). In patients with GN, anti–α‐actinin, but not anti‐dsDNA, antibodies correlated with the SLEDAI score (minus the anti‐DNA component) and with treatment. The fraction of serum anti‐dsDNA antibodies that cross‐reacted with α‐actinin exhibited high avidity for dsDNA, as determined using a commercial EIA for high‐avidity anti‐dsDNA antibodies and an in‐house conventional ELISA.

Conclusion

The α‐actinin–binding antibodies are significantly associated with GN in SLE. Whether such autoantibodies may anticipate the development of this complication of SLE remains to be verified.

     

Cross-reactivity of human lupus anti-DNA antibodies with alpha-actinin and nephritogenic potential

OBJECTIVE: Cross-reactivity with kidney antigens is believed to be a critical determinant in the renal pathogenicity of anti-double-stranded DNA (anti-dsDNA) antibodies. Murine nephritogenic anti-dsDNA antibodies have been shown to cross-react with alpha-actinin, and anti-alpha-actinin antibodies have been found to be deposited in the kidneys of lupus mice with active nephritis. Furthermore, in humans with systemic lupus erythematosus (SLE), it has been found that a greater proportion of polyclonal IgG anti-dsDNA antibodies from patients with renal involvement bind to alpha-actinin than do those from patients without renal disease. We undertook this study to substantiate a direct link between cross-reactive anti-dsDNA/anti-alpha-actinin antibodies and the pathogenesis of lupus nephritis in humans. METHODS: A panel of 10 anti-dsDNA and/or anti-alpha-actinin antibodies was generated by Epstein-Barr virus transformation of lymphocytes from patients with SLE and was extensively characterized. Antibody binding was studied by enzyme-linked immunosorbent assay and Western blotting. Antibody potential for pathogenicity was assessed by measuring binding to isolated glomeruli and mesangial cells and by evaluation of histologic features of the kidney following injection in vivo. RESULTS: All anti-dsDNA antibodies isolated also bound alpha-actinin. Cross-reactive antibodies bound to mesangial cells and to isolated glomeruli ex vivo. Binding to glomeruli was not inhibited by DNase treatment, but could be abrogated by alpha-actinin. Furthermore, histopathologic abnormalities seen in mice injected intraperitoneally with a cross-reactive cell line included fusion of podocyte foot processes and subepithelial and subendothelial deposition. CONCLUSION: These studies provide strong support for the hypothesis that alpha-actinin is a major cross-reactive target for anti-dsDNA antibodies in SLE patients. Cross-reactive anti-dsDNA/anti-alpha-actinin antibodies from SLE patients are pathogenic and may contribute to the kidney lesions in lupus nephritis.     

Antigenic triggers and molecular targets for anti-double-stranded DNA antibodies

While anti-double-stranded (ds)DNA antibodies are a characteristic serologic hallmark for SLE, the triggering antigen is unknown. Using phage display libraries, we identified DWEYSVWLSN as a peptide mimic of DNA for a pathogenic anti-dsDNA antibody. Peptide immunization of non-autoimmune mice induced anti-dsDNA as well as other lupus-associated antibodies. Molecular analysis of the induced anti-dsDNA antibodies revealed several similarities with anti-dsDNA antibodies that appear spontaneously in lupus mice. Furthermore, lupus-prone mice immunized with this peptide DNA mimic had higher autoantibody titers as well as more severe nephritis. Anti-DNA antibodies may contribute to lupus nephritis via cross-reactivity with renal antigen. Using western blotting of lysates of mesangial cells from a lupus mouse, we found that a pathogenic anti-DNA antibody binds to alpha-actinin. High titers of anti-alpha-actinin antibodies were present in the sera and kidney eluates of lupus mice with active disease. Binding to alpha-actinin was diminished in mesangial cells derived from BALB/c mice, suggesting that target antigen expression may play a role in determining autoantibody binding to the kidney. We conclude that a pathogenic, lupus-like autoantibody response can be induced by a peptide antigen, and that alpha-actinin is a cross-reactive renal target for the pathogenic anti-dsDNA autoantibody response in lupus mice.     

CLINICAL SIGNIFICANCE OF ANTI–DOUBLE-STRANDED DNA ANTIBODIES DETECTED BY A SOLID PHASE ENZYME IMMUNOASSAY

A solid phase enzyme immunoassay (EIA) detected anti-double-stranded (ds) DNA antibodies in 88% of sera from patients classified clinically as having active systemic lupus erythematosus (SLE) without renal symptoms and 93% with renal disease. Fifty-six percent of sera from patients with inactive SLE were EIA positive for anti-dsDNA antibodies. The EIA had a sensitivity and specificity comparable to radioimmunoassay (RIA) and hemagglutination for patients with active SLE with or without renal disease, but it detected anti-dsDNA antibodies more frequently in patients with inactive SLE than the latter procedures. Precipitating antibodies detected by counterimmunoelectrophoresis (CIE) were less common in patients with renal disease (23% incidence) than clinically active patients without renal disease (79% incidence). Twenty-four SLE sera with elevated levels of CIq binding showed a 96% concordance for a positive EIA for anti-dsDNA antibodies in contrast to 66% concordance by RIA or hemagglutination. These findings suggest that the EIA is a sensitive and specific method for detection and measurement of anti-dsDNA antibodies. Several clinical applications of the EIA are discussed.     

Anti-nucleosome antibody: significance in lupus patients lacking anti-double-stranded DNA antibody

OBJECTIVE: To investigate the clinical significance of anti-nucleosome antibodies in SLE patients lacking anti-double stranded DNA (dsDNA) antibodies. METHODS: IgG anti-nucleosome antibodies were detected by enzyme-linked immunosorbent assays (ELISA) in the sera of SLE patients. Anti-dsDNA antibodies were measured by Farr assays and ELISA, not only in the samples taken for anti-nucleosome testing, but also in sera obtained regularly during the follow-up. RESULTS: Ninety-eight (76.0%) out of 129 patients with SLE had anti-nucleosome antibodies. Twenty-five patients (19.4%) consistently showed little or no anti-dsDNA reactivity during the course of their disease, and among these anti-nucleosome antibodies were present in the sera of 15 (60.0%). Of the patients with anti-dsDNA-negative SLE, renal disorders were present in 8 patients (32.0%), all of whom had anti-nucleosome antibodies. Renal disorders were not found in patients (n = 10) who had neither anti-dsDNA nor anti-nucleosome antibodies. Other autoantibodies such as anti-Ro, anti-Sm and anti-cardiolipin were not associated with renal disorders in this group. The levels of anti-nucleosome antibody strongly correlated with the SLEDAI scores, but inversely correlated with serum complement levels in anti-dsDNA negative SLE patients. CONCLUSION: Our data suggest that the anti-nucleosome antibody may be a useful marker for diagnosis and activity assessment of anti-dsDNA negative SLE. Anti-nucleosome antibody may be an important factor for renal involvement in this subgroup of patients.     

Restricted specificity of anti-ribosomal P antibodies to SLE patients in Israel

OBJECTIVE: Anti-ribosomal P antibodies (aRib-P Ab) are highly specific for systemic lupus erythematosus (SLE), but their correlatation with disease activity and manifestations including renal, hepatic and central nervous system (CNS) involvement is still controversial. The aim of our study was to evaluate the prevalence of aRib-P Ab and their correlation with clinical manifestations and anti-dsDNA antibodies in SLE patients from Israel. METHODS: Elevated titers of aRib-P Ab utilizing the ELISA method were analyzed in 141 sera samples from 44 SLE patients, 20 Familial Mediterranean Fever (FMF) patients, 22 primary antiphospholipid syndrome (PAPS) patients, 12 patients with infections, and 43 healthy individuals. The SLEDAI score was utilized for assessing SLE disease activity. RESULTS: Elevated titers of aRib-P Ab were present in 11% of SLE patients (n = 6). The mean SLEDAI was 7 (range: 3-10). No statistically significant association was observed between the presence of aRib-P Ab and disease manifestations present in the SLEDAI. The 6 SLE patients had renal disease (n = 1), leucopenia (n = 1), rash (n = 3), and CNS involvement manifested as psychosis (n = 1) or depression (n = 1). Elevated titers of anti-dsDNA antibodies were found in 50% of patients with elevated titers of aRib- P Ab. Patients with PAPS, FMF, infections or healthy controls did not harbor elevated titers of aRib-P Ab. CONCLUSION: Elevated titers of aRib-P Ab were restricted to SLE patients. We confirm previously reported associations of aRib-P Ab reactivity with disease activity and elevated anti-dsDNA Ab titers. No significant correlation with a specific manifestation described on the SLEDAI score was established in this small cohort of patients.     

Use of a new fluorescence immunoassay to detect anti-dsDNA antibodies is more correlated with disease activity and complement than the ELISA method in SLE patients

To determine whether the serum levels of anti-double strand DNA (anti-dsDNA) autoantibodies detected using a newly developed fluorescence immunoassay (FIA) in patients with systemic lupus erythematosus (SLE) correlated more with clinical parameters, such as SLE disease activity index (SLEDAI), complement and the occurrence of nephritis when compared with traditional enzyme-linked immunosorbent assay (ELISA), we prospectively collected 124 serum samples from 31 patients who had juvenile-onset SLE and were regularly monitored every 2 months at our outpatient clinic. At every visit, clinical manifestations and laboratory parameters were assessed and the SLEDAI was determined. Correlation analyses between the two different measurements of anti-dsDNA antibodies and SLEDAI, serum complement levels and the occurrence of nephritis were performed. The results showed that anti-dsDNA autoantibodies detected using both ELISA and FIA significantly correlated with SLEDAI, and significantly and inversely correlated with the serum levels of C3 and C4. FIA had significantly higher correlation with SLEDAI and C4 than did ELISA. The mean values of anti-dsDNA antibodies detected using FIA in patients with nephritis were significantly higher than in those without nephritis. In contrast, the values of anti-dsDNA antibodies detected using ELISA did not show significant differences between these two groups. We conclude that FIA had better correlation with SLEDAI, C4 and the occurrence of nephritis, and comparable correlations with C3 that were similar to the results found using ELISA. Thus, it is worthwhile developing the FIA method for clinical evaluation of disease activity in SLE patients.     

Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity

BACKGROUND: The presence of "anti-DNA antibodies in abnormal titres" is a well established criterion for SLE classification, but there is no agreement on the performance of this test. OBJECTIVE: To study the correlation between clinical findings and five different solid and solution phase anti-DNA antibody assays. METHODS: 158 consecutively collected ANA positive sera were studied in a double blind fashion. Anti-DNA antibodies were determined by different solid phase assays (ssDNA-, dsDNA- specific ELISA, EliA anti-dsDNA assay, Crithidia luciliae assay), and by an experimental solution phase anti-DNA assay using biotinylated pUC18 plasmid, human, calf thymus, and E coli DNA. Antibody affinity was determined by surface plasmon resonance. Clinical data were obtained independently of the laboratory analyses and later related to the anti-dsDNA findings. RESULTS: Anti-dsDNA antibodies were most frequently detected by ELISA, but were not specific for SLE as they were present in up to 30% of other disease groups. Those detected by the Crithidia luciliae assay were predictive for SLE, while antibodies binding in solution phase ELISA using the pUC18 correlated strongly with the Crithidia luciliae assay. Surface plasmon resonance analysis showed that antibody binding to pUC18 was not due to higher relative affinity for dsDNA in general, but apparently to specificity for that plasmid DNA. Serum samples from three patients with lupus nephritis were positive in both pUC18 solution phase and Crithidia luciliae assays. CONCLUSIONS: Assay principle selection is decisive for the detection of clinically significant anti-DNA antibodies. Revision of the anti-DNA antibody criterion in the SLE classification may be needed.     

Critical comparative analyses of anti-alpha-actinin and glomerulus-bound antibodies in human and murine lupus nephritis

OBJECTIVE: Although anti-double-stranded DNA (anti-dsDNA) antibodies are important in lupus nephritis, the question regarding which glomerular structures (alpha-actinin, nucleosomes, or others) are recognized by nephritogenic anti-dsDNA antibodies is still controversial. In this study, we determined which glomerular structures are recognized by monoclonal and in vivo-bound nephritogenic antibodies. METHODS: Western blotting was used to analyze the ability of nephritogenic anti-dsDNA antibodies to recognize glomerular and nucleosomal structures. Sera from patients with lupus nephritis, sera from random antinuclear antibody-positive patients, and paired antibodies from sera and kidney eluates from nephritic (NZB x NZW)F(1) mice were analyzed for activity against proteins identified by monoclonal nephritogenic antibodies, and against alpha-actinin, dsDNA, nucleosomes, histone H1, heparan sulfate, DNase I, and type IV collagen. Immunoelectron microscopy was used to determine the glomerular localization of alpha-actinin and in vivo-bound autoantibodies in nephritic (NZB x NZW)F1 mouse kidneys. RESULTS: Anti-alpha-actinin antibodies were observed in human and murine lupus nephritis sera and in sera from patients without systemic lupus erythematosus and were not detected in kidney eluates from nephritic mice. Antibodies to dsDNA and histone H1 were detected in all eluates. Western blot analyses revealed that nephritogenic anti-dsDNA antibodies recognized a 32-kd band, identified as histone H1. Competitive enzyme-linked immunosorbent assay demonstrated that nephritogenic monoclonal antibodies, and dominant antibodies eluted from nephritic kidneys, cross-reacted with dsDNA and H1. This cross-reactive anti-H1 specificity was largely absent in sera from those mice. Immunoelectron microscopic analysis of nephritic (NZB x NZW)F1 mouse kidneys revealed that antibodies eluted from kidneys, but not anti-alpha-actinin antibodies, bound to distinct nephritis-associated electron-dense structures linked to glomerular basement membranes. CONCLUSION: Cross-reactive anti-dsDNA/anti-histone H1 antibodies, but not anti-alpha-actinin antibodies, are central among those deposited in nephritic glomeruli.     

Anti-dsDNA antibody avidity determination by a simple reliable ELISA method for SLE diagnosis and monitoring

High avidity anti-dsDNA antibodies are more specific for SLE diagnosis, and more closely associated with renal involvement than intermediate or low-affinity anti-dsDNA antibodies. ELISA methods are largely used to detect anti-dsDNA, but their high sensitivity is inversely related to specificity because they also detect low avidity antibodies. We developed an ELISA assay based on the law of mass action and the competitive binding of dsDNA in solution and coated to microwells with anti-dsDNA antibodies. A simplified Scatchard plot analysis system was used to measure anti-dsDNA antibody avidity which was expressed as apparent affinity constant (Kaa), and quantified in liters per unit (I/U). We prospectively studied 101 consecutive SLE patients, who were followed for 3 years; three serum samples were sequentially collected from each patient during follow-up for determination of IgG anti-dsDNA antibody concentration, and anti-dsDNA avidity. SLE disease activity was estimated using the European Consensus Lupus Activity Measure (ECLAM) index. Sera from 100 healthy subjects and 133 patients with other connective tissue diseases or infectious diseases were also assayed as controls. The mean Kaa in SLE patients was 65.2 +/- 47.3 l/U, with no variations over time. Anti-dsDNA-positive SLE patients had higher Kaa values (79.1 +/- 46.8) than anti-dsDNA negative patients (27.2 +/- 20.1; P < 0.001). No correlation emerged between anti-dsDNA avidity and the ECLAM activity index score. Avidity was significantly higher in patients with renal involvement vs patients without this complication (78.2 +/- 50 vs 59.9 +/- 45.6 l/U; P = 0.0013). This simple ELISA method could be very useful in the diagnostic phase to differentiate high avidity anti-dsDNA autoantibodies that are characteristically found in SLE patients from low avidity antibodies that can also be found in other inflammatory diseases. Moreover, our data confirm the predictive value of high avidity anti-dsDNA antibodies for the development of lupus nephritis.     

Antibodies to DNA in patients with systemic lupus erythematosus. Their role in the diagnosis, the follow-up and the pathogenesis of the disease

In this paper an overview of the present knowledge on antibodies against DNA will be presented. Diagnostic, prognostic and pathogenic aspects of anti-DNA will be highlighted. Detection of antibodies to DNA in the circulation of a patient by an assay selective for high avidity anti-DNA is highly diagnostic for SLE. Anti-DNA of low avidity occurs in rheumatic diseases other than SLE as well, making detection of such antibodies of less diagnostic value. Furthermore, data will be presented that show that the anti-DNA ELISA in its present form is not suited as a diagnostic tool. Not only disease features of SLE vary from patient to patient, anti-DNA avidity does so too. A relationship between anti-DNA avidity and clinical features can be found: high avidity anti-DNA is more abundant in patients with nephritis, low avidity anti-DNA in patients with CNS involvement. Prognostically, a steady increase of the level of high avidity anti-DNA generally heralds an upcoming exacerbation in a patient. Furthermore, 85% of the patients who do not have SLE at the time (high avidity) anti-DNA is detected in their serum, will develop the disease within the next few years. It is noteworthy, that patients with only low avidity anti-DNA in their circulation develop a more mild form of SLE; the (low) avidity of their anti-DNA seldomly increases during the course of their disease. The relevance of anti-DNA to the pathogenesis of SLE still is a matter of debate. On the one hand, the association of parameters of anti-DNA that determine the size of the complexes formed with DNA is in favour of the classical hypothesis, which states that SLE is primarily an immune complex disease. On the other hand, recent data on crossreactions of anti-DNA with phospholipids, glycosaminoglycans and other (polynegative) structures plead for a role of such crossreactivities in the pathogenesis of SLE.     

Specificity analysis of antibodies to single-stranded micrococcal DNA in the sera of normal human subjects and patients with systemic lupus erythematosus.

To evaluate the properties of antibodies to bacterial DNA in the sera of normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE), the effects of ionic strength and pH on their binding to single-stranded DNA (ssDNA) from Micrococcus lysodeikticus (MC) were measured. By ELISA, antibodies to MC ssDNA in NHS showed greater activity at high ionic strength (0.2-1.0 M) than antibodies in lupus sera. Similarly, antibodies in NHS had higher activity at pH 9 than lupus anti-DNA. Competition binding assays indicated, moreover, that NHS anti-DNA showed greater inhibition by DNA than lupus anti-DNA at comparable inhibitor concentrations. Together, these results suggest that antibodies to MC ssDNA in NHS and SLE sera may differ in their mode of interaction with bacterial DNA and that NHS can generate high avidity antibodies to at least certain DNA determinants.     

Anti-serum amyloid component P antibodies in patients with systemic lupus erythematosus correlate with disease activity

OBJECTIVE: To determine the presence of raised titres of anti-serum amyloid P component (SAP) antibodies in patients with systemic lupus erythematosus (SLE) and to evaluate their correlation with clinical disease by the SLEDAI and clinical manifestations. METHODS: 452 samples were screened for raised anti-SAP antibody titres by an ELISA. Clinical measures and SLEDAI scores were independently reviewed from medical records. 21 serial samples from 7 patients with SLE were assessed for a change in anti-SAP antibody titres after treatment. RESULTS: Raised anti-SAP antibody titres were detected in 145/328 (44%) SLE samples. In 112 randomly selected samples, 69/112 (62%) patients had raised anti-SAP antibodies and anti-dsDNA antibody titres, whereas only 32/112 (28%) had raised anti-dsDNA antibody titres without raised anti-SAP antibody titres. The mean titre of anti-SAP antibodies in patients with active disease was higher than in patients with inactive disease and controls. SLEDAI scores, assessed in 54 patients, were raised in 26/31 (84%) patients with raised anti-SAP antibody titres. A SLEDAI score >or=8 was found in 16/31 (52%) patients with raised anti-SAP antibody titres but in only 5/23 (22%) patients without raised titres. No specific pattern of disease was detected in patients with or without raised titres of anti-SAP antibodies. Serial sampling from patients with active SLE and raised anti-SAP antibody titres showed that anti-SAP antibody titres decreased after treatment and correlated with clinical improvement. CONCLUSION: Raised anti-SAP antibody titres detected in patients with SLE correlate with disease activity and decrease with improvement of clinical disease, and thus may serve as an additional prognostic marker.     

ANTI-DNA ANTIBODIES IN THE PRIMARY ANTIPHOSPHOLIPID SYNDROME (PAPS)

Primary antiphospholipid syndrome (PAPS) is considered a distinctentity from SLE and patients with PAPS are generally regardedas being dsDNA antibody negative. Levels of IgG and IgM ss andds DNA antibodies were measured by ELISA in 30 patients whofulfilled the criteria for the diagnosis of PAPS. We comparedthese patients with 20 normal controls and seven patients withidiopathic SLE. We also examined all the sera for anti-nuclearantibodies by Hep-2 cells and for dsDNA antibodies by Crithidia. We found that 16 patients with PAPS had antibodies to ss and/ordsDNA. Only three of the 16 positive patients had both IgG andIgM anti-DNA antibodies. Twelve patients had anti-nuclear antibodies,but only two were weakly positive for dsDNA antibodies by Crithidiaimmunofluorescence. Eleven out of 30 patients with PAPS hadIgM anti-dsDNA antibodies compared to two out of the seven SLEpatients. The PAPS patients with anti-DNA antibodies were clinicallyindistinguishable from the PAPS patients without antibodiesagainst DNA. Our results show that 53% of patients with PAPS had antibodiesto DNA which supports the view that PAPS and SLE are probablyoverlapping disorders. KEY WORDS: ELISA, Primary antiphospholipid syndrome, Anti-DNA antibodies     

Anti-dsDNA: choice of assay in relation to clinical value

Summary Antibodies to DNA are quite specific for systemic lupus erythematosus (SLE) and occur in the majority of SLE patients. Therefore, their detection is an important diagnostic aid to the clinician. Detection of anti-dsDNA may precede the diagnosis of SLE by more than a year. Fluctuations in the level of anti-dsDNA in an individual patient may give important information on the clinical status of the patient. Four of the most important methods developed for the measurement of anti-dsDNA antibodies will be discussed in this paper: the Farr assay, the PEG assay, the indirect immunofluorescence test on Crithidia luciliae and the ELISA. They will also be compared with one commercially available (Farr) assay, the Amersham anti-dsDNA kit. Each method, detects a part of the spectrum of anti-dsDNA antibodies produced by a patient. The Farr assay is the most specific for SLE; however, milder forms of the disease in which patients have only low avidity anti-dsDNA may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related more frequently to the occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are found more often in patients with central nervous system involvement. Traditionally, SLE is considered an immune-complex disease, in which inflammatory processes are initiated by local deposition of DNA/anti-dsDNA complexes. More recently, a major role was thought to be played by crossreactions of anti-dsDNA with tissue constituents. Our current view, however, is that such a crossreactivity plays only a minor role; we postulate that binding to glomerular constituents is caused by anti-dsDNA antibodies complexed with DNA and histones.     

Association of IgA anti-dsDNA antibodies with vasculitis and disease activity in systemic lupus erythematosus

Previously it has been suggested that the presence of antibodies against dsDNA of the IgA class may define a subset of systemic lupus erythematosus (SLE) patients suffering from nephritis and arthritis. Therefore, these autoantibodies were measured in sera of 352 patients with SLE, 81 blood donors, and 189 patients with rheumatoid arthritis using a new ELISA based on human recombinant dsDNA as antigen. IgA anti-dsDNA antibodies were found in 19.9% of the sera from patients with SLE, but in none of the sera from 81 normal controls and 189 patients with rheumatoid arthritis. The association of these autoantibodies with 31 clinical and 36 laboratory parameters was calculated. IgA anti-dsDNA antibodies were found to be associated with parameters of disease activity such as elevated erythrocyte sedimentation rate and consumption of complement component C3, and the clinical parameters vasculitis, acral necrosis and erythema, but not with nephritis and arthritis. Therefore, IgA anti-dsDNA antibodies define a subset of SLE patients, and monitoring of IgA anti-dsDNA antibodies may be helpful as a prognostic parameter in patients with SLE. Received: 12 April 1998 / Accepted: 24 April 1998     

Correlations between antinucleosome antibodies and anti-double-stranded DNA antibodies, C3, C4, and clinical activity in lupus patients

OBJECTIVE: To examine the correlations between antinucleosome antibodies and anti-double-stranded (ds) DNA antibodies, complement (C) 3 and 4 levels, and clinical activities in SLE patients. METHODS: Antinucleosome antibodies and anti-dsDNA antibodies were detected by enzyme-linked immunosorbent assays (ELISA). The levels of C3 and C4 were measured by nephelometry. Clinical activities were determined by SLE Disease Activity Index (SLEDAI). RESULTS: Of 65 SLE patients, the prevalence of antinucleosome antibodies were higher than anti-ds DNA antibodies (52.3 vs 36.9%, respectively, p < 0.05). Similar results were obtained in 45 active SLE patients, 64.4% for antinucleosome antibodies and 46.7% for anti-ds DNA antibodies. Of 34 patients lacking anti-ds DNA antibodies, 16 (47.1%) were shown antinucleosome antibodies. Activity of antinucleosome antibodies was significantly correlated with the SLEDAI scores and inversedly correlated with the C3 levels but not with the C4 levels. CONCLUSION: Antinucleosome antibodies could be one of the earliest and most sensitive markers in diagnosis of SLE, particularly in anti-dsDNA antibodies-negative patients. More importantly, antinucleosome antibodies is correlated with clinical activities and C3 levels.     

Anti-dsDNA antibody assay: high specificity and sensitivity with a filtration radioassay in comparison to low specificity with the standard ELISA

OBJECTIVE: To evaluate whether a new fluid-phase filtration radioassay possesses both high sensitivity and specificity compared with the currently used ELISA and Farr assays. METHODS: Sequential sera (25 samples) from 9 patients with systemic lupus erythematosus (SLE), sera from 20 patients with SLE possessing anti-dsDNA antibodies by the Crithidia assay, 75 patients with rheumatoid arthritis possessing rheumatoid factors, 50 healthy control subjects, 767 from patients with type 1 diabetes, and a commercial standard serum sample were tested for anti-dsDNA antibodies with the 3 different assays. RESULTS: Of serial dilutions of a standard anti-dsDNA antibody sample, only the highest positive sample (50 IU/ml) in the ELISA and the highest 2 positive samples (50 and 25 IU/ml) in the Farr assay were above the normal range. In contrast, all dilutions (to 2.5 IU/ml) of the standard anti-dsDNA antibody sample were above the normal range in the filtration radioassay. Using the values of 50 healthy control subjects in each assay to define the normal range, all 25 sequential sera from 9 patients with SLE were positive. In addition, 20/20 of the SLE individual sera, 2/75 (2.7%) of the RA sera, and 12/767 (1.6%) of the diabetes sera were positive (signal above normal range) in the filtration radioassay. The SLE sera were further examined in 2 additional assays, ELISA and Farr assay, and both assays were less sensitive and specific compared with the filtration radioassay. CONCLUSION: The fluid-phase filtration radioassay demonstrated high sensitivity and specificity for the detection of anti-dsDNA antibodies in SLE, with the standard ELISA exhibiting lower specificity. We suggest that testing for anti-dsDNA antibodies can be improved using a fluid-phase filtration radioassay in comparison to commercial assays.     

Evidence for direct anti-heparin-sulphate reactivity in sera of SLE patients

Recently it has been suggested that anti-dsDNA antibodies (Abs) promote tissue damage in systemic lupus erythematosus (SLE) by cross-reactivity with highly negatively charged tissue components such as heparan sulphate (HS), the major glycosaminoglycan of the glomerular basement membrane (GBM). Other authors, however, support the theory of DNA-anti-dsDNA immune complex deposition in situ. To further elucidate the possible role of HS antibodies, we developed a new ELISA system with heparan sulphate bound to solid phase. SLE patients (n=40) showed a higher reactivity against HS (mean=28.4, SD=34.3) as compared to normal donors (n=28, mean=15.2, SD=6.3) and patients with rheumatoid arthritis (n=35, mean=14.3, SD=6.4). The addition of native dsDNA or HS to SLE sera was followed by a dose-dependent reduction in anti-HS reactivity. In contrast, in an anti-dsDNA ELISA, no reduction was observed when HS was added to SLE sera. An increase in reactivity was observed when SLE sera with and without a prior incubation with dsDNA were digested with DNAse I or II. After the purification of serum samples by protein A sepharose under dissociative conditions, seven out of eight SLE patients showed an increase in anti-HS reactivity. No correlation of the anti-HS Abs was found with organ involvement or other serological parameters. We concluded, that there is evidence for a direct anti-HS Ab reactivity in SLE sera. A part of these antibodies seems to show low avidity anti-dsDNA cross-reactivity.