Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.     

Autoantibodies to the Ro/SSA antigen are conformation dependent. I: Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous and the autoanti-Ro/SSA response is correspondingly heterogeneous. There are two isoform families; the 60 kD forms and the 52 kD forms. We studied the antigenic difference between the native and denatured Ro/SSA isoforms and found that the autoanti-Ro/SSA response to the native 60 kD antigen is quite homogeneous. All anti-Ro/SSA sera recognize the native kD antigen regardless of the reactivities to the 60 kD band on the Western blot. Surprisingly, no anti-Ro/SSA sera without anti-La/SSB reacts with the native 52 kD Ro/SSA, although sera with both precipitating anti-Ro/SSA and anti-La/SSB can immunoprecipitate the native 52 kD antigen. Anti-Ro/SSA sera exist which react exclusively with the native 60 kD Ro/SSA protein (10/43, 23%) while no anti-Ro/SSA sera have been found which react exclusively with the denatured 52 kD Ro/SSA antigen. In sera with anti-Ro/SSA precipitins alone, only antibody to the denatured 52 kD Ro/SSA molecule is found! In sera with anti-Ro/SSA and anti-U1 RNP precipitins, no antibody to either native or denatured 52 kD Ro/SSA is found, while in sera with both anti-Ro/SSA and anti-La/SSB precipitins, antibodies to both the native and denatured forms of 52 kD Ro/SSA are present. These data suggest that the anti-Ro/SSA response to the 60 kD molecule is driven by the native 60 kD Ro/SSA molecule while the molecular identification of the antigen drive in the anti-52 kD Ro/SSA response is unknown.     

Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein.

Patients suffering from systemic lupus erythematosus (SLE) or Sjögren's syndrome (SS) often contain autoantibodies directed to the Ro(SS-A) complex. In this study the antigenic determinants on two of the components of the Ro complex, i.e. the Ro60 and the Ro52 polypeptides, were investigated. Anti-Ro+ sera were selected by counter-immunoelectrophoresis. Depending on the detection method, 59-68% of the SLE patients produced anti-Ro but not anti-La antibody, while 72-81% of the SS patients produced both anti-Ro and anti-La antibody. Immunoprecipitation of recombinant Ro-proteins showed that 61 sera (87%) were reactive with both Ro proteins, seven sera with Ro60 only, one serum with Ro52 only, and one serum did not precipitate the proteins at all. The anti-Ro60 reactivity of human sera is strongly associated with the native form of Ro60, suggesting that conformational autoepitopes are an important feature of Ro60. In the case of Ro52, frequently the residues located between amino acids 216 and 292 were essential for reactivity with the antibodies. With 70% of the lupus sera tested this appeared to be the only region important for reactivity. The antibodies of SS patients generally recognized multiple B cell epitopes located between amino acids 55 and 292. The results of this study indicate that the antigenic determinants on Ro52 are different for autoantibodies produced by lupus patients compared with those of SS patients.     

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren''s syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren''s syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren''s syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.     

IgM and IgG Subclass Distribution of Human Anti-Ro/SSA 60 kDa Autoantibodies

The Ro/SSA and La/SSB antigens are common targets for autoantibodies found in the sera of patients with Sjögren's syndrome and SLE. The anti-Ro/SSA and anti-La/SSB antibodies often appear together but are not cross-reactive. This paper describes the humoral autoimmune response to the Ro/SSA 60 kDa protein moiety with respect to the presence of IgM and IgG1-4 antibodies. IgM antibodies to the Ro 60 kDa protein coexisted with IgG anti-Ro 60 kDa antibodies in nearly half of the sera. A similar fraction also contained IgM anti-La/SSB antibodies. The frequency of sera with IgM antibodies of both specificities was that expected from random overlap.
A predominating igGl anti-Ro 60 kDa response was found in all patients, but anti-Ro 60 kDa antibodies of the other IgG subclasses were present also in a high number of sera. This is in contrast to the reported IgG subclass distribution of anti-La/SSB antibodies.
Mapping of IgM and IgG 1–4 antibody recognition of different parts of the Ro 60 kDa protein was also performed. IgM and IgGl-4 antibodies of all sera reacted with the central part of the Ro 60 kDa protein, encompassing amino acid residues 181–320.     

Human Anti-Ro Autoantibodies Bind Peptides Accessible to the Surface of the Native Ro Autoantigen

The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60kDa Ro are variably bound by anti-Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti-Ro precipitin negative lupus patients. The specific activities of anti-peptide antibodies and of anti-native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity-enriched anti-native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti-Ro positive sera. Two peptides (residues 274–290 and 480–494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity-enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti-Ro octapeptide autoantibodies are part of the population of anti-native Ro autoantibodies in this naturally arising autoimmune response.     

A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: Sequence similarity with a viral protein

The Ro ribonucleoprotein is composed of hY RNA and a 60.7-kD peptide that is antigenic for autoantibodies produced by many patients with systemic lupus erythematosus or Sjögren's syndrome and mothers of newborns with complete congenital heart block. A major immunoreactive fragment (13 kD) of the 60-kD Ro is bound by 28 of 45 (62%) of the anti-Ro sera tested. Amino acid sequence analysis localizes this fragment to the carboxyl end of the 60-kD Ro peptide. All possible overlapping octapeptides of this 13-kD peptide of 60-kD Ro have been assessed for antigenicity. Sera that bind the 13-kD peptide fragment in immunoblot generally also bind the octapeptides of Ro spanning the sequence AIALREYRKKMDIPA (P<0.01). Inhibition studies with synthetic peptides and purified Ro have established specificity for reference serum antibody binding to an antigenic octapeptide, EYRKKMDI, from this region. The closely related sequence EYRKKLMD is found in the nucleocapsid protein of vesicular stomatitis virus and may portend an immunologic link to this or a related viral antigen. These results also demonstrate that despite fine specificity variation between human sera, there are recurring patterns of anti-Ro binding shared by some patients who have precipitating anti-Ro autoantibodies.     

Heterogeneity of the Ro/SSA antigen and autoanti-Ro/SSA response: evidence of the four antigenically distinct forms.

Precipitating antibodies to the Ro/SSA antigen occur in the sera of 40% of patients with sytemic lupus erythematosus (SLE) and in 40–70% of the sera of patients with primary Sjögren's syndrome. Previous work has shown that lymphocyte extracts contain two Ro/SSA antigens with protein moieties of 60 kD and 52 kD and that erythrocyte haemolysate contain two analogous but antigenically distinct Ro/SSA molecules of 60 kD and 54 kD. Frequency analysis of the various specificities in 43 sera with precipitating anti-Ro/SSA and studies with affinity-eluted antibodies suggest that the lymphocyte 60 kD and erythrocyte 60 kD Ro/SSA molecules are related as are the lymphocyte 52 kD and erythrocyte 54 kD Ro/SSA proteins. Anti-Ro/SSA sera when accompanied by other precipitins (anti-La/SSB and anti-U1RNP) react preferentially with certain Ro/SSA isoforms. Evidence is also presented for a 45 kD form of Ro/SSA. These data suggest that the antigenic heterogeneity of the Ro/SSA antigen is immunologically relevant and that there are two families of Ro/SSA antigens: one comprising of the two 60-kD proteins in the erythrocyte and lymphocyte and the other the erythrocyte 54 kD and lymphocyte 52 kD Ro/SSA proteins, respectively.     

Autoantibodies to the Ro/SSA autoantigen are conformation dependent. II: Antibodies to the denatured form of 52 kD Ro/SSA are a cross reacting subset of antibodies to the native 60 kD Ro/SSA molecule.

The immune response to the Ro/SSA particles is conformation dependent. In sera with only anti-Ro/SSA precipitins, the autoantibodies to the 60 kD Ro/SSA are largely to the native 60 kD Ro/SSA while autoantibodies to the 52 kD Ro/SSA particle when present are exclusively to the denatured 52 kD Ro/SSA particle. Antibodies eluted from a recombinant 52 kD Ro/SSA fusion protein reacted in a sandwich ELISA which only measures antibody to native 60 kD Ro/SSA antigens and this reaction is largely inhibited by native homogeneous 60 kD Ro/SSA. In addition, antibody binding to the 52 kD Ro/SSA antigen in Western blot is also strongly inhibited by native 60 kD Ro/SSA. These experiment strongly suggest that reactivity of denatured 52 kD Ro/SSA antigen represents a cross reaction with autoantibodies directed to the native 60 kD Ro/SSA antigen. As a corollary of these experiments, data are presented that suggest the hY-RNAs are not associated with the 52 kD Ro/SSA protein but only with the 60 kD Ro/SSA protein. These data are consistent with the hypothesis that the autoanti-Ro/SSA response is driven by native 60 kD Ro/SSA and the immune response to denatured 52 kD Ro/SSA is largely a cross-reactive subset of the immune response to native 60 kD Ro/SSA.     

Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy

We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52⁺ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1⁺ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti-Ro52⁺ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long α-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1⁺ and anti-Jo-1⁺IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.     

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.     

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen,enhancing recognition by anti-Ro60 kD autoantibodies

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide–calreticulin is recognized specifically by anti‐Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi‐step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175–184aa(10p) and 216–232aa(17p). The interaction of the peptide–calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty‐eight anti‐Ro60 KD positive and 23 anti‐Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti‐Ro60 kD positive sera bound the complex calreticulin‐17p, while 95% of the same sera had activity against the complex calreticulin ? 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti‐Ro60 kD positive sera. Anti‐Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10ρ and 17ρ or with the complexes calreticulin ? 10p and calreticulin‐17p (<5%). These results suggest that calreticulin can induce conformation‐dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.     

Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52?kDa Ro, 60?kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sjögren's syndrome (SS). The differences between 52?kDa, 60?kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52 kDa Ro, 60 kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sj?gren's syndrome (SS). The differences between 52 kDa, 60 kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

The detection of anti-Ro/SS-A and anti-La/SS-B antibodies. A comparison of counterimmunoelectrophoresis with immunoblot, ELISA, and RNA-precipitation assays.

The presence in serum of anti-Ro/SS-A and/or anti-La/SS-B autoantibodies is a characteristic of autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus. To evaluate different assays currently available for the detection of these antibodies 50 sera were tested using the four different assay methods: counterimmunoelectrophoresis (CIE), RNA precipitation assay, immunoblotting technique and ELISA. The RNA-precipitation assay showed the highest sensitivity and specificity. The CIE for the detection of anti-Ro/SS-A antibodies gave comparable results whereas the Ro/SS-A ELISA and Ro/SS-A or HeLa immunoblot showed lower sensitivities (96% and 80% respectively). Sensitivity was even lower (66%) when only reactivity towards the 60 kDa Ro/SS-A protein was considered. The ELISA for the detection of anti-La/SS-B antibodies showed a sensitivity of 98%, the immunoblotting technique of 86% and the CIE only 67%. The high sensitivity of the La/SS-B ELISA went together with a low specificity of 14%. We conclude from these data that for the detection of anti-Ro/SS-A and anti-La/SS-B antibodies the RNA precipitation assay shows the highest sensitivity and highest specificity. For routine screening purposes the CIE is the most convenient and reliable assay to detect anti-Ro/SS-A antibodies. For the detection of anti-La/SS-B antibodies the immunoblot corresponds most closely to the RNA precipitation assay.     

Clinical significance of anti-Ro52 (TRIM21) antibodies non-associated with anti-SSA 60kDa antibodies: results of a multicentric study

Ro52 antigen has recently been identified as TRIM21 protein, but the clinical significance of anti-Ro52/TRIM21 antibodies remains controversial. The aim of this multicentric study was to investigate the significance of anti-Ro52 antibodies without anti-SSA/Ro60 antibodies in various connective diseases. Sera were selected by each laboratory using its own method (ELISA, immunodot or Luminex technology), and then performed with ANA Screen BioPlex? reagent (BIO-RAD). Among the 247 screened sera, 155/247 (63%) were confirmed as anti-Ro52 positive and anti-SSA/Ro60 negative. These sera were analyzed for the detection of other antibodies in relation with clinical settings. Isolated anti-Ro52 antibodies were detected in 89/155 (57%) sera. For the remaining sera (66/155), the main antibodies associations were Sm/SmRNP or Chromatin (n=38; 57%), Jo1 (n=17; 26%) and CenpB (n=9; 14%). Clinical data from the 155 patients showed high prevalence in autoimmune diseases (73%) including myositis or dermatomyositis (n=30), lupus (n=23); Sj?gren and/or sicca syndrome (n=27); CREST or Systemic sclerosis (n=11) and autoimmune hepatitis (n=11). We found that pulmonary manifestations were often associated with the presence of anti-Ro52 antibodies (n=34, 22%), in addition with anti-tRNA synthetases, anti-SRP or anti-Ku antibodies (18/34) or isolated in half of cases (16/34). Separate detection of anti-Ro52 antibodies might be useful in related antisynthetase syndrome diagnosis. The presence of anti-Ro52 antibodies should probably precede development of autoimmune disease and must induce sequential follow-up of positive patients, particularly in interstitial lung disease progression.     

Sjögren's syndrome: autoantibodies to cellular antigens. Clinical and molecular aspects

Autoantibodies to cellular autoantigens are usually found in sera of patients with systemic autoimmune rheumatic diseases. Patients with Sj?gren's syndrome (SS) frequently present autoantibodies to both organ and non-organ-specific autoantigens. The most commonly detected autoantibodies are those directed against the ribonucleoproteins Ro/SSA and La/SSB. The presence of the antibodies in SS is associated with early disease onset, longer disease duration, parotid gland enlargement, higher frequency of extraglandular manifestations and more intense lymphocytic infiltration of the minor salivary glands. Over the past several years, the structure and function of these autoantigens have been extensively studied. Several centers, using different techniques, have investigated the B cell epitopes on the protein components Ro 60 kD, Ro 52kD, and La 48 kD. Finally, increased evidence of direct involvement of anti-Ro/SSA and anti-La/SSB autoantibodies in the pathogenesis of tissue injury has been contributed by several studies.     

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.     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术（IIF）抗核抗体（ANA）检测法的底物HEp-2细胞，建立抗60kDRo60／SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA，克隆入真核表达载体pEGFP-C1，并转染HEp-2细胞。通过荧光显微镜、免疫印迹法（IBT）及IIF鉴定转染细胞（HEp-Ro60）中Ro60-绿色荧光蛋白（GFP）融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro／SSA对流免疫电泳（CIE）检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达，融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6．7倍（P〈0．01），而且2例HEp-2细胞上IIF检测为阴性的血清在HEI-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体，并增加了ANA检出的敏感性。     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术(IIF)抗核抗体(ANA)检测法的底物HEp-2细胞,建立抗60kDRo60/SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA,克隆入真核表达载体pEGFP-C1,并转染HEp-2细胞。通过荧光显微镜、免疫印迹法(IBT)及IIF鉴定转染细胞(HEp-Ro60)中Ro60-绿色荧光蛋白(GFP)融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro/SSA对流免疫电泳(CIE)检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达,融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6.7倍(P<0.01),而且2例HEp-2细胞上IIF检测为阴性的血清在HEp-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体,并增加了ANA检出的敏感性。