Lupus-derived autoantibodies with dual autoactivity: anti-DNA and anti-Fc. I. Comparison of IgG autoreactivities with single-chain Fv derivatives.

Investigations into the intrinsic affinity and reactivity of autoanti-DNA active sites were initiated through the use of purified monoclonal IgG and the synthesis of single-chain Fv derivatives of murine monoclonal anti-DNA autoantibodies BV 04-01 and BV 17-45. Results showed that relative to the respective IgG hybridomas, only the BV 04-01 SCA derivative showed demonstrable reactivity with DNA. The monovalent single-chain derivative of BV 17-45 showed no reactivity with DNA in solution or solid-phase assays, even though the parental IgG had been previously described as high affinity. However, 17-45 displayed reactivity as a bivalent single-chain derivative. In addition, upon concentration, BV 17-45 IgG formed a highly stable, papain-resistant precipitate. Investigations into the nature of the precipitate revealed that BV 17-45 possessed significant, DNA-inhibitable autobinding to its own IgG molecule. BV 04-01 also possessed similar anti-self reactivity. Thus, both monoclonal autoantibodies examined in this study possessed dual binding specificity; anti-DNa and anti-self.     

Production and analysis of IgG monoclonal anti-DNA antibodies from systemic lupus erythematosus (SLE) patients.

This study compares recently devised methods for producing IgG anti-DNA MoAbs from patients with SLE and analyses the antibodies generated from one patient at different phases of disease. Lymphocytes from SLE patients were transformed with Epstein-Barr virus(EBV) and/or fused with a heteromyeloma cell line, CB-F7. Direct fusion with CB-F7 resulted in the highest proportion of IgG-secreting lines, whereas EBV transformation resulted in a high percentage of IgM-secreting lines. Using direct fusion, five IgM anti-DNA antibody-secreting hybridomas were generated using lymphocytes from a patient with relatively inactive SLE. Six months later when the disease was active, only IgG anti-DNA antibodies were produced. The antigen-binding patterns of the MoAbs were analysed. Only one of the IgM anti-DNA antibodies reacted with dsDNA by ELISA and none by Crithidia immunofluorescence, whereas two of the IgG antibodies reacted with dsDNA by ELISA and Crithidia but did not bind to ssDNA. Only the two IgG high affinity anti-dsDNA antibodies bound to histones, and this was enhanced by added DNA, whereas three IgM antibodies bound to cardiolipin. This study supports the notion that MoAbs derived from a patient with SLE represent those found in the serum of SLE patients at different stages of disease activity. The binding to histones by the two IgG anti-dsDNA antibodies supports the recently expressed view that antibodies binding DNA/histone may be important in the pathogenesis of SLE.     

Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization

Extracellular calreticulin (CRT) as well as anti‐CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in ‘epitope spreading’ to other autoantigens such as the Ro/SS‐A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti‐CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjögren’s syndrome. Approximately 40% of all SLE patients were positive for anti‐CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1–289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N‐terminal half of the protein in 69% of the SLE sera from active disease patients, while the C‐domain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P‐domains. Sera from both healthy and disease controls and primary Sjögren’s syndrome patients were non‐reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N‐terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.     

BXSB小鼠血清抗DNA抗体水平及其对淋巴细胞转化的影响

用酶联免疫吸附剂测定法（ＥＬＩＳＡ）和３Ｈ－胸腺嘧啶核苷掺入法，观察一种自发性系统性红斑狼疮（ＳＬＥ）好发小鼠（ＢＸＳＢ小鼠）在不同月龄时，血中抗ＤＮＡ抗体水平的变化，并观察该血清对正常ＬＡＣＡ小鼠淋巴细胞转化的影响。结果显示，自３月龄起，ＢＸＳＢ雄性鼠血中的抗ＤＮＡ抗体水平开始升高，４月龄上升至高峰，５月龄仍保持较高水平，各月龄ＢＸＳＢ血清与对照血清相比，Ｐ＜０．０５（ＡＮＯＶＡ）。同时，该血清对正常ＬＡＣＡ小鼠淋巴细胞转化也有抑制作用，以４月龄血清的抑制作用最大。如果将同一只小鼠血清抗ＤＮＡ抗体水平的变化与该血清对淋巴细胞转化的影响做回归和相关分析，发现两者有明显的正相关关系（，０．２３２，＜一５３，尸＜Ｏ．０５）。提示雄性＊ｘ８Ｂ小鼠可能从３月龄开始表现ＳＬＥ。发病后，血清中产生某种能够抑制淋巴细胞转化的物质，它的产生与抗ＤＮＡ抗体有平行关系。     

In vitro induction of IgG anti-DNA antibody from high density B cells of systemic lupus erythematosus patients by an HLA DR-restricted T cell clone.

An HLA-DR restricted T cell clone (26G11) which recognized a lymphoid cell-derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti-DNA antibody production in DR-matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR-matched normal control subjects produced large amounts of IgM anti-DNA antibody, but did not produce IgG anti-DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR-matched patients with active SLE spontaneously produced a fairly large amount of IgG anti-DNA antibody, and the production was augmented by the T cell clone. Little IgG anti-DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD-B) and high density B (HD-B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti-DNA antibody was spontaneously produced by LD-B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti-DNA antibody was not spontaneously produced by the HD-B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti-DNA antibody-producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production.     

The role of cpg sequences in the induction of anti-DNA antibodies

C1 esterase inhibitor (C1INH) is an important regulatory protein of the classical pathway of complement. Mutations in the gene for this protein cause the autosomal dominant disorder hereditary angioedema (HAE). Approximately 85% of patients with HAE have a Type I defect, characterized by a diminished level of antigenic and functional C1INH. Patients with Type II defects have sufficient protein, but one allele produces dysfunctional protein. We have sequenced the DNA from HAE patients and have discovered four previously unreported mutations. The first mutation is a splice site error at nucleotide 8721, which changes the 3' acceptor splice site AG to GG at the end of intron 5 at nucleotide 8721-8722. The second mutation is a single base insertion in exon 3 between nucleotides 2467 and 2468. The third mutation is a missense error present in the eighth exon of the C1INH; at nucleotide 16867 (amino acid 470), a T to A mutation transforms a Met to a Lys. The fourth mutation closely resembles the third mutation in that it is a missense error occurring in exon 8 in the distal hinge region; a T16827C substitution changes the Phe at amino acid 457 to Leu. This report compiles a list of 97 distinct defects in the C1INH gene that cause hereditary angioedema.     

DNA and Heparin Alter the Internalization Process of Anti-DNA Monoclonal Antibodies According to Patterns Typical of Both the Charged Molecule and the Antibody

The internalization into CHO-K1 fibroblasts of three polyreactive monoclonal IgG2a anti-DNA autoantibodies (mAbs), F14.6, J20.8 and F4.1, isolated from the same unimmunized (NZBxNZW) F1 mouse, and synthetic peptides derived from F4.1 was studied using a technique which quantifies nuclear accumulation. The localization of the mAbs was intranuclear. We compared the influence of two negatively-charged molecules, DNA or heparin. At low concentrations, DNA had dual effects-inhibitory or stimulatory-depending on the mAb. Heparin was inhibitory or had no effect. The possibility that proteoglycans are 'receptors' recognized by anti-DNA mAbs which bind through heparin-sensitive reactions, was explored. Only F4.1 internalization was partly inhibited in glycosaminoglycan-deficient cells. We propose that the complex alterations of internalization patterns of these polyreactive mAbs by the two negatively charged molecules can be explained by (a) the potential of polyreactive mAbs to bind to various charge (or conformation-) dependent 'receptors', (b) the potential of a subclass of mAbs complexed with DNA to utilize additional 'receptor(s)'. Glycosaminoglycans were required for internalization of F4.1-derived peptides, which remained extranuclear, suggesting that nuclear internalization of mAb F4.1 is a multistep process that requires certain sequences present on the intact mAb.     

系统性红斑狼疮患者自身抗体检测及意义

目的：探讨自身抗体在系统性红斑狼疮（SLE）中的意义。方法：采用放射免疫分析检测50例SLE患者血清中的抗双链DNA（dsDNA）抗体,采用德国IMTEC公司抗核抗体谱线性印迹法检测抗核小体抗体（AnuA）、抗组蛋白抗体（AHA）、抗StuD1等其他自身抗体。结果：抗StuD1阳性率最高（82％）,其次是抗R060KD抗体（80％）和AunA（72％）;抗dsDNA、AHA、抗U1-RNP、抗R052KD、抗SSB的阳性率分别为44％、32％、58％、48％、24％。其他三种自身抗体阳性率很低。结论：SLE患者血清中可出现大量的自身抗体,同时检测多种自身抗体能提高对SLE的鉴别诊断。     

Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process.

Anti-single stranded DNA (ssDNA) and anti-double stranded DNA (dsDNA) B cells are regulated in non-autoimmune mice. In this report we show that while both anti-ssDNA and anti-dsDNA B cells are blocked in their ability to differentiate into antibody-secreting cells, other phenotypic and functional characteristics distinguish them from one another. Splenic anti-ssDNA B cells are found distributed throughout the B cell follicle, and are phenotypically mature and long-lived. On the other hand, splenic anti-dsDNA B cells are short-lived, exhibit an immature and antigen-experienced phenotype, and localize to the T-B interface of the splenic follicle. Functionally, anti-ssDNA B cells proliferate, albeit suboptimally, in response to anti-IgM, lipopolysaccharide (LPS) and CD40L/IL-4 + anti-IgM stimulation, and tyrosine phosphorylate intracellular proteins upon mIgM cross-linking. Anti-dsDNA B cells, on the other hand, are functionally unresponsive to anti-IgM and LPS stimulation, and do not phosphorylate intracellular proteins, including Syk, upon mIg stimulation. Importantly, anti-DNA B cell anergy is maintained in the absence of T cells since both anti-ssDNA and anti-dsDNA B cells are as efficiently regulated in RAG2(-/-) mice as in their RAG2(+/+) counterparts. Interestingly, the severely anergic state of anti-dsDNA B cells is partially reversible upon stimulation with CD40 ligand and IL-4. In response to these signals, anti-dsDNA B cells remain viable, up-regulate cell surface expression of B7-2 and IgM, and restore their ability to proliferate and phosphorylate Syk upon mIg cross-linking. Collectively, these data suggest that anti-DNA B cell anergy encompasses distinct phenotypes which, even in its most severe form, may be reversible upon stimulation with T cell-derived factors.     

DNA Antibody Idiotypes: An Analysis of Their Clinical Connections and Origins

Approximately thirty common DNA antibody idiotypes have been described on hybridoma derived or affinity purified DNA-binding antibodies. There are associations between some idiotypes and the clinical manifestations of systemic lupus erythematosus although none are sufficiently firm to be clinically useful in identifying subsets of SLE or in assessing disease activity in individual patients. The expression of these idiotypes is not confined to DNA antibodies in SLE. They may be found in the serum from patients with a range of autoimmune rheumatic disorders, infectious diseases and blood dyscrasias. In most cases the antigen binding specificity of the antibody bearing the idiotype is unknown. The precise relationship between the various idiotypes is becoming better understood with increasing availability of genetic and structural data. DNA antibody idiotype manipulation may provide a potential new therapeutic modality in SLE.     

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab)₂ fragments. Family members' sera had higher binding to anti-DNA F(ab)₂ and to normal IgG F(ab)₂ fragments (P<0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab)₂ than to normal IgG F(ab)₂ fragments (P<0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The anti-idiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.     

Epstein-Barr virus-transformed B cells bearing idiotypes of anti-DNA autoantibodies

Epstein-Barr virus (EBV)-transformed B cells obtained from healthy subjects had the same idiotypes of anti-DNA autoantibodies on their surface as those obtained from patients suffering from systemic lupus erythematosus. These clones secreted anti-single-stranded or antidouble-stranded DNA antibodies. Among them, some produced anti-DNA idiotype-positive antibodies but failed to bind DNA. This was confirmed by a competitive inhibition radioimmunoassay. It was then considered whether or not the expression of anti-DNA idiotype on B-cell clones related to the anti-DNA antibody activityin vivo. The amounts of anti-DNA antibodies were not associated with the incidence of idiotype-positive B cells in the EBV-transformed cell lines from normals. The results indicate that the clones committed to the synthesis of anti-DNA idiotype-positive antibodies commonly exist at a resting state in the circulation of healthy subjects, probably through the self-tolerance regulatory system.     

IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus

We describe the production of six mouse-human heterohybridomas secreting human IgG anti-dsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of anti-DNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay. Inhibition studies revealed a remarkable specificity for certain polynucleotide structures. To our knowledge these are the first hybridomas described in the human system that secrete anti-dsDNA antibodies of the IgG class.     

Comparative study of catalytic (DNA-hydrolyzing) and cytotoxic properties of anti-dna autoantibodies

DNA-hydrolyzing and cytotoxic properties of anti-DNA autoantibodies isolated from patients with systemic autoimmune diseases and from autoimmune MRL-lpr/lpr, SJL/J, and (NZB× NZW)F₁ mice were studied. Cytotoxic and catalytic properties of these antibodies correlated. A relationship between the stage of systemic lupus erythematosus and catalytic and cytotoxic properties of DNA abzymes was revealed. Of all studied cells, L929 cells were most sensitive were most sensitive to in vitro effect of antibodies. Treatment of target cells with anti-DNA autoantibodies with cytotoxic properties induced internucleosomal DNA fragmentation, which is characteristic of apoptosis.     

In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies

Anti-nuclear autoantibodies (ANA) are the hallmark of systemic autoimmune diseases. Yet, the in vivo function of ANA remains controversial to a large extent due to the intracellular nature of their antigenic targets. It has been reported that a subset of autoantibodies can penetrate live cells and translocate into the subcellular compartments containing the corresponding antigens. The studies presented herein show that murine anti-Sm and anti-La monoclonal autoantibodies can also enter a variety of cell types from different animal species and that the cell penetration activity is not isotype-restricted. Interestingly, only mAb with cross-reactivity against double-stranded DNA did enter cells. Both these autoantibodies rapidly accumulate in the nucleus of viable cells but display different penetration kinetics. In co-localization experiments, monoclonal autoantibodies did not accumulate significantly within endocytic vesicles containing dextran, suggesting that they are internalized by mechanisms distinct from conventional receptor-mediated endocytosis. This report represents the first evidence that anti-La and anti-Sm autoantibodies are capable of entering live cells. Our observations support the notion that the phenomenon of intracellular autoantibodies may have a larger scope than previously reported and are consistent with a potential pathogenic role for ANA.     

Anti-heparan sulphate reactivity in sera from patients with systemic lupus erythematosus with renal or non-renal manifestations.

Previously, we have shown that anti-DNA can bind to heparan sulphate (HS), a constituent of the glomerular basement membrane (GBM). We hypothesized that binding of anti-DNA to HS in the GBM plays a role in the onset of systemic lupus erythematosus (SLE) nephritis. To test this hypothesis we measured the anti-HS reactivity in cross-sectional and longitudinal studies of SLE patients with or without nephritis. In the transverse serum study single serum samples from 26 SLE patients were studied. We found no correlation between anti-HS reactivity and previously development of nephritis (anti-HS positive: seven out of 16 with history of nephritis, two out of 10 without nephritis). However, six of the seven anti-HS positive sera in the nephritis group were obtained within 1 month of the onset of nephritis, suggesting a temporal relationship between anti-HS reactivity and onset of nephritis. In the longitudinal serum study between six and 16 serum samples were studied from each of 10 SLE-patients. In five out of five episodes of nephritis we found anti-HS reactivity before the onset or exacerbation of the nephritis. In four non-renal manifestations anti-HS reactivity was found in only one episode; in none of the three patients who remained clinically stable did serum samples show anti-HS reactivity. Anti-HS reactivity was only found in sera positive for anti-DNA by Farr assay but the anti-HS titre was not a mere reflection of the reactivity measured in the Farr assay. This indicates that only a subpopulation of anti-DNA can bind to HS. We found a high correlation (r = 0.99) between anti-HS reactivities in plasma and serum and we conclude that anti-HS reactivity in serum samples from SLE patients is not due to in vitro complex formation during clotting. Although further prospective analysis is necessary, our data suggest that measurement of anti-HS reactivity in SLE patients might identify patients at risk for the development of nephritis.     

Anti-Ro fine specificity defined by multiple antigenic peptides identifies components of tertiary epitopes

Anti-Ro (or SSA) is a clinically important autoantibody that is found in 25–40% of patients with systemic lupus erythematosus as well as an even greater proportion of patients with Sjögren's syndrome or subacute cutaneous lupus. We have studied the binding of anti-Ro sera to multiple antigenic peptides constructed from the sequence of the 60-kD Ro molecule. The results demonstrate that sera bind these peptides in solid-phase assay. Surprisingly, some of these peptides also form a precipitin line in double immunodiffusion with anti-Ro sera. Formation of lines of identity in double immunodiffusion as well as absorption studies indicate that peptides distant in the primary amino acid sequence and without shared sequence are bound by the same antibody. In addition, data from surface plasmon resonance demonstrate that peptides identified in this manner have protein–protein interactions. Thus, these techniques may identify the components of conformational epitopes.     

False-negative anti-DNA antibody activity in infantile systemic Lupus erythematosus (SLE)

A 2.5-month-old previously healthy female infant presented with serositis, nephrotic syndrome, progressive renal failure, anemia, and thrombocytopenia. Renal biopsy revealed a proliferative glomerulonephritis with glomerular and extraglomerular deposits of IgG, IgM, C3, and Cl_q by direct immunofluorescence (IF) techniques. Skin biopsy was positive for IgG and C3 deposits in the dermal-epidermal junction by IF. Despite strong clinical and pathological criteria for systemic lupus erythematosus (SLE), tests for antinuclear and anti-DNA antibodies were negative. Circulating immune complexes (CICs) were detected in three separate assay systems. Immunochemical analysis of isolated CICs showed that anti-DNA antibody was present. Analysis of kidney biopsy material by antigen-specific solubilization techniques showed antibodies reactive with ds-DNA in the kidney. These studies confirm that SLE may be a cause of the congenital nephrotic syndrome and that negative SLE serologies may be secondary to binding of available antibody by excess antigen. Analysis of CICs may be helpful in confirming the diagnosis of SLE in seronegative patients.     

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

The B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145–164, 289–308, 301–320 and 349–368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes:¹⁴⁷HKAFKGSI¹⁵⁴, ²⁹¹NGNLQLRNKEVT³⁰², ³⁰¹VTWEVLEGEVEKEALKKI³¹⁸ and ³⁴⁹GSGKGKVQFQGKKTKF³⁶⁴. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presented 83.3% similarity with the ¹³⁹HKGFKGVD¹⁴⁶ region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope ¹⁴⁷HKAFKGSI¹⁵⁴ to 100% to epitope ³⁴⁹GSGKGKVQGKKTKF³⁶⁴. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.     

Reactive oxygen species modify human DNA, eliciting a more discriminating antigen for the diagnosis of systemic lupus erythematosus.

During the development of an ELISA to measure anti-DNA antibodies in systemic lupus erythematosus (SLE) sera, native dsDNA was found not to be the most appropriate antigen to use in ELISA assays for differentiating between SLE patients and those with rheumatoid arthritis (RA), a disease also associated with circulating serum anti-DNA antibodies. By modifying the ELISA technique to incorporate human DNA, denatured by reactive oxygen species, to detect anti-DNA antibodies in SLE sera, results consistently showed an increase in antibody binding when compared with the native antigen; no such trend was observed in the comparable group of RA patients. Using this assay serum anti-dsDNA antibody levels were measured in a group of 20 controls, 20 RA patients (10 seropositive and 10 seronegative) and 30 SLE patients (15 with clinically active disease, 15 with inactive disease). A comparison with the standard radioimmunoassay used to measure anti-DNA antibodies for the diagnosis of SLE showed that the ELISA assay using modified DNA performed better than the standard radioimmunoassay offering an improvement in both clinical specificity and sensitivity. The improved method particularly reduced the problem of false-negative results for SLE patients shown clinically to be either mildly active or inactive.