Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases

BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.     

New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sj?gren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases

Background: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay.

Methods: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested.

Results: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92–97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively.

Conclusion: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.     

Detection, epitope-mapping and function of anti-Fas autoantibody in patients with silicosis

Dysregulation of apoptosis through the Fas-Fas ligand pathway is associated with the onset of autoimmune disease. Since autoantibodies directed against unknown antigens are present in the sera of these patients, sera samples were examined for the presence of autoantibodies directed against the Fas molecule. Using Western blotting and a ProteinChip analysis, autoantibodies against Fas were detected in patients with silicosis, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and weakly detected in healthy individuals. Using epitope mapping employing 12-amino-acid polypeptides with the SPOTs system, a minimum of four epitopes and a maximum of 10 epitopes were found. Several amino acid residues involved in binding FasL, such as C66, R87, L90, E93 and H126, were presented within the epitopes. Serum containing a large amount of anti-Fas autoantibody from silicosis patients inhibited the growth of a Fas-expressing human cell line, but did not inhibit the growth of a low Fas-expresser nor a Fas-expresser in which the Fas gene had been silenced by small interference RNA. All epitopes in the intracellular region of Fas were located in the death domain. The possible roles of anti-Fas autoantibody detected in healthy volunteers and patients with silicosis or autoimmune diseases are discussed here.     

New Coupled-Particle Light-Scattering Assay for Detection of Ro/SSA (52 and 60 Kilodaltons) and La/SSB Autoantibodies in Connective Tissue Diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

Onset and regulation of anti-lamin B autoantibody production is independent of the level of polyclonal activation

Anti-lamin B autoantibodies are associated with both systemic lupus erythematosus (SLE) and autoimmune liver disease. We examined the possibility that the underlying clinical feature in patients with anti-lamin B autoantibodies might be chronic autoimmune liver disease, and whether the hypergammaglobulinemia present in both disorders is involved in generating anti-lamin B autoantibodies. A lamin B fusion protein (MLB1), consisting of amino acids 77-533 of lamin B fused to TrpE, was used to screen sera from 84 patients with SLE for anti-lamin B autoantibodies. 3/4 prototype human lamin B antisera, 5/84 SLE sera (6%), and 0/30 sera from healthy individuals reacted with MLB1 on immunoblots at a 1:500 dilution. Of the 9 anti-lamin B autoantibody positive patients studied, all but 1 fulfilled at least four ARA criteria for SLE. None of the patients displayed evidence of chronic autoimmune liver disease, suggesting that autoimmune liver disease is not strongly associated with anti-lamin B antibodies in SLE. In SLE, as in "lupoid hepatitis", anti-lamin B autoantibodies are often produced transiently during periods of increased disease activity. Although polyclonal hypergammaglobulinemia is also associated with increased activity of both diseases, anti-lamin B autoantibody production in 2 patients was independent of total immunoglobulin levels, antibodies to irrelevant proteins, and production of some other autoantibodies. Thus, polyclonal activation is insufficient to explain either the initiation or regulation of anti-lamin B autoantibody production, supporting the hypothesis that antinuclear antibodies are antigen-selective.     

Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. The SLE Study Group.

Recent studies have shown that sera of patients suffering from systemic autoimmune diseases contain autoantibodies directed against the eukaryotic ribosomal protein L7 [1]. In the present study we screened a large panel of sera from patients with systemic lupus erythematosus (SLE) for the presence of anti-L7 autoantibodies and their relationship to clinical, serological and genetic parameters of SLE. By means of an ELISA employing recombinant protein L7 as antigen we detected anti-L7 autoantobodies in 172 of 506 SLE sera (34%). Negative correlations were observed between the presence of anti-L7 autoantibodies, serum IgG levels and proteinuria; a potentially positive relationship existed with lung fibrosis. In order to analyse further this possibility we screened sera of 129 patients suffering from progressive systemic sclerosis (PSS) for anti-L7 reactivity; 45 of these patients had lung fibrosis. Of the PSS patients, 41% exhibited anti-L7 autoantibodies, but positive reactions were evenly distributed among patients with and without lung fibrosis. Protein L7 thus represents a major autoantigen of systemic autoimmune diseases, but does not so far define a distinct subpopulation of patients.     

Development of an ELISA for the detection of autoantibodies to BP230

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the transmembrane hemidesmosomal protein BP180/collagen type XVII and the intracellular plaque protein BP230. The aim of the present study was to develop an ELISA system for the detection of circulating autoantibodies to BP230. We generated five overlapping cDNA constructs covering the entire length of BP230 and expressed them in baculovirus-infected Sf21 insect cells. ELISA reactivity against BP230 was found in 63% of 56 BP patients' sera; the specificity of the ELISA was 93%. Epitope mapping studies showed that the fragment representing the C-terminal portion of BP230 was by far the most frequent target within the molecule. This ELISA provides a useful tool for the detection of autoantibodies to BP230 in BP and other diseases associated with an autoimmune response to this protein.     

Comparison of immunoperoxidase staining with indirect immunofluorescence, ELISA, and Western blotting assays for detecting anti-HTLV-I antibodies in systemic lupus erythematosus.

Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based on the Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five various autoimmune diseases with one or more circulating autoantibodies. Serum samples from three patients were found to be HTLV-I-positive by the ELISA assay and sera from five patients showed a non-specific reaction by indirect immunofluorescence. These sera were negative when tested by indirect immunoperoxidase staining and Western blotting assay. All four methods gave positive results when tested with samples from 19 HTLV-I carriers and 16 patients with adult T cell leukaemia. Indirect immunoperoxidase staining and Western blotting assay are probably useful and more specific assays for the detection of anti-HTLV-I antibodies in samples from patients with autoimmune diseases.     

Intramolecular epitope spreading among anti-caspase-8 autoantibodies in patients with silicosis,systemic sclerosis and systemic lupus erythematosus,as well as in healthy individuals

Dysregulation of apoptosis through the Fas-Fas ligand pathway is relevant in autoimmune disease onset. We recently reported elevated serum levels of sFas in patients with silicosis, systemic sclerosis (SSC) and systemic lupus erythematosus (SLE), and proposed a block of apoptosis in the pathogenesis. The disturbance of apoptosis in lymphocytes including autoreactive clones could induce autoantibody production. Since autoantibodies directed against unknown antigens are present in the sera of these patients, the sera samples were examined for the presence of autoantibodies directed to caspase-8. Using Western blotting, autoantibodies against caspase-8 were detected in healthy individuals and in over 60% of patients. Using epitope mapping employing 12 amino acid polypeptides with SPOTs system, a minimum of 4 epitopes and a maximum of 13 were found, which implied that epitope spreading was in progress. It is noteworthy that two important catalytic cystein residues were included within the epitopes; firstly the active site cystein Cys287, and secondly Cys360 located in the unique pentapeptide motif QACQG. Using recombinant human caspase-8 linked protein chip array, autoantibodies were identified and molecular weight determined. The antibodies were mainly IgG; 80% were subclass IgG1(lambda); 20% were IgG4(kappa). Despite the ratio of human light chain kappa:lambda = 2:1, the predominance of IgG1(lambda) is noticeable. Anti-caspase-8 autoantibodies are detectable in healthy individuals and in patients suffering silicosis, SSc or SLE. A few epitopes were detected in healthy individuals compared to those suffering autoimmune diseases, indicating the intramolecular epitope spreading. Relationship of autoantibodies and the clinical background of the patients requires clarification.     

Recent advances in diagnostic technologies for autoimmune diseases

The investigation of autoimmunity provides an interest challenge in "omics" research and, particularly, proteome research, as autoimmune diseases are common disorders of unsolved etiology that occur in a wide range of manifestations, in all of which tissues and organs are attacked by the body's own immune system. Autoantibodies are a hallmark of many autoimmune diseases and the presence of autoantibodies is a distinctive and key characteristic of autoimmune diseases. Conventionally, the study of autoimmune response has always been conducted by analysing the presence and/or concentration of individual antibodies in biological fluids. New proteomic techniques allow the simultaneous identification/measurement of different autoantibodies in sera of patients suffering from autoimmune diseases. The possibility of simultaneously measuring a number of correlated analytes appears to be very interesting for analytical reasons (reduced volumes of biological samples, reagents and low costs), logistical/managerial reasons, and pathophysiological reasons (combination of markers in disease-oriented or organ-oriented profiling). In particular, we describe data collected by using high-throughput techniques such as antigen microarrays and mass spectrometry for antibody profiling. While recently collected data demonstrate satisfactory analytical sensitivity and reproducibility, some issues such as standardization and data interpretation have to be solved before the introduction of these new and promising techniques into clinical practice.     

Galactosylation of serum IgG and autoantibodies in murine models of autoimmune haemolytic anaemia.

A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the CH2 domain oligosaccharide. The current aim was to determine whether the galactosylation of serum IgG is also reduced in a classic antibody-mediated, organ-specific autoimmune condition, and whether the pathogenic autoantibodies are preferentially G0. In two murine forms of autoimmune haemolytic anaemia (AIHA), sera and autoantibodies eluted from erythrocytes were obtained, and the levels of G0 measured using a lectin-binding assay. Serum IgG galactosylation was unaffected following the induction of AIHA in CBA/Igb mice by immunization with rat erythrocytes, but in all animals with the disease the IgG autoantibodies generated were more G0 than the sera. The anti-rat erythrocyte antibodies were similar to the autoantibodies in being preferentially G0, and when CBA/Igb mice were immunized with canine erythrocytes as a control foreign antigen, there was again a bias towards the production of G0 IgG antibodies. In NZB mice with chronic, spontaneous AIHA, the concentration and galactosylation of both serum IgG and autoantibodies were lower than in the induced model, and the ratio of G0 IgG in the serum and erythrocyte eluates varied markedly between different individuals. Our interpretation of these results is that changes in serum IgG or autoantibody galactosylation are not consistent in different models of AIHA, and that production of low galactosyl antibodies can be a feature of a normal immune response.     

Autoantibody production against the U small nuclear ribonucleoprotein particle proteins E, F and G in patients with connective tissue diseases

The nucleoplasmic U small nuclear ribonucleoprotein particles (snRNP) have a set of seven proteins in common which are designated B', B, D, D', E, F and G. Patients suffering from rheumatoid autoimmune diseases such as systemic lupus erythematosus often develop autoantibodies against the proteins B', B, and D. Here we describe a sensitive immunoassay which allows the specific detection of autoantibodies reacting with the E, F or G snRNP proteins. We were able to identify several patient sera containing autoantibodies against one or more of these proteins. This demonstrates that all snRNP proteins described so far are potentially antigenic in systemic rheumatoid diseases. The characterization of the antibodies showed an immunological cross-reactivity between the snRNP protein G and the 70-kDa protein of U1 snRNP. Several sera contained autoantibodies which were specific for the F snRNP protein.     

Comparing HEp-2 cell line with rat liver in routine screening test for antinuclear and antinucleolar autoantibodies in autoimmune diseases

The comparative study of the human tumor cell line HEp-2 and rat liver for the detection of antinuclear and antinucleolar autoantibodies by the indirect immunofluorescence technic in routine screening test demonstrate that, taking titer and staining pattern into account, both substrates are able to separate autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, scleroderma and primary biliary cirrhosis) patients from healthy subjects. The minimal screening test must include sera diluted 1:20 and 1:80. The capability of the HEp-2 substrate to reveal and to discriminate different speckled nuclear and nucleolar patterns explain its greater performance, notably in detecting anticentromere antibodies highly specific for the CREST syndrome and a speckled nuclear antibody frequently associated with primary biliary cirrhosis, allowing an earlier diagnosis of autoimmune diseases presenting these patterns.     

Absence of autoimmune serological reactions in chronic non A, non B viral hepatitis.

In 18 cases of chronic liver disease due to non-A, non-B hepatitis virus(es) in which the diagnosis was established by transmission, including chimpanzee inoculation in nine, sera were tested for the autoantibodies characteristically associated with autoimmune chronic active hepatitis. The frequency of autoantibodies to nuclear, smooth muscle, cytofilament, mitochondrial and liver membrane antigens was low, being not greater than that recorded for a normal population, and the few positive reactions obtained were at very low titre. These findings suggest that among cases of 'HBsAg negative' chronic hepatitis, those due to NANB infection are distinguishable from those due to autoimmune chronic hepatitis by negative serological tests for autoantibodies.     

Clinical features of anti-chromo antibodies associated with anti-centromere antibodies

Anti-chromo antibodies (AChA) are autoantibodies accompanying anti-centromere antibodies (ACA). We determined the frequency and clinical significance of AChA in autoimmune rheumatic diseases. Serum samples from 252 patients with rheumatic diseases were examined by immunoblotting with HeLa nuclear extract and with recombinant N-terminus of 25-kD chromo protein (p25). AChA were detected in 28 (36%) of 77 sera with ACA. AChA were found only in ACA-positive sera. Twenty-two (79%) of 28 recognized a recombinant N-terminal portion of p25, including the chromo domain which is conserved among species. AChA were related to leucopenia, thrombocytopenia, elevated erythrocyte sedimentation rate, and existence of Sjögren*s syndrome (SS). In ACA-positive patients, AChA might be a serologic indicator of systemic sclerosis (SSc), having features of systemic lupus erythematosus and/or SS or diseases other than SSc.     

Autoantibodies against small nucleolar ribonucleoprotein complexes and their clinical associations

Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA-protein complexes, the so-called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynaud's phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin-positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti-Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co-diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.     

Detection of anti-brain serum antibodies using a semi-quantitative immunohistological method

The number of autoimmune disorders that may involve the nervous system is increasing. The diagnosis of neurological involvement in the context of systemic diseases may be helped by the detection of autoantibodies reacting against neural autoantigens. If the autoantigen is not known but the target tissue is suspected, immunohistochemistry is one of the main techniques used to certify the presence of autoantibodies. Autoreactive antibodies are also present in the healthy population but in low quantity compared to patients with such diseases. Quantification of such autoantibodies could help to discriminate between disease and healthy states. We have developed a densitometric immunohistological method for the evaluation of human serum anti-neural reactivity. Using a densitometric analysis of rat brain sections incubated with the serum from 107 healthy subjects, we have defined the baseline of natural anti-neural autoreactivity, and the cut-off for subsequent quantification of anti-neural reactivity in patients with neurological involvement in the context of autoimmune diseases, including systemic lupus erythematosus, paraneoplastic cerebellar degeneration, and stiff person syndrome. The test sensitivity was 81% with a positive predictive value of 52%, a specificity of 89% with a negative predictive value as high as 97%. In conclusion, this standardised semi-quantitative procedure makes immunohistochemistry a reliable diagnostic test for autoimmune neuropathologies and represents an excellent exclusion test for anti-neural autoimmunity.     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.