Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

Detection of anti-early endosome antigen 1 antibody in sera showing cytoplasmic vesicular staining,taken from patients with connective tissue diseases

An early endosome antigen previously reported by F.T. Mu could be stained on cytoplasmic vesicles of HEp-2 cells. Here, we have investigated autoantibodies against cytoplasmic vesicular antigens, especially against early endosome antigen 1. Twelve sera were selected on the basis of cytoplasmic vesicular staining patterns of HEp-2 cells. Protein-immunoprecipitation using 35S-methionine labeled HeLa lysates, and RNA immunoprecipitation using 32P-labeled HeLa lysates were conducted to characterize the cognate antigens. Nine of 12 sera reacted with proteins in the range of 162-180 kDa, three of which were found to react specifically with the 162 kDa 35S methionine labeled recombinant early endosome antigen 1. These proteins were not associated with common RNA. Although complete clinical information was not available, some of the patients had rheumatoid arthritis(RA). In addition, the RNA-IPP results suggest that other patients included one each with SLE, SSc, polymyositis, and Sj?gren's syndrome. Anti-early endosome antigen 1 antibody was found in 25%(3 of 12) of sera known to stain cytoplasmic vesicles. The reactive sera came mostly from patients with RA. The sera was from one case each of clinical-confirmed RA, SLE and Sj?gren's syndrome.     

重组人Jo-1自身抗原的克隆、原核表达及抗原特异性鉴定

目的:旨在获得高纯人Jo-1抗原(即组氨酰-tRNA合成酶)。方法:利用RT-PCR从人胎盘总RNA中获得Jo-1的编码基因,并分别用IMPACT-CN和pMAL系统的pTYB11、pMAL-c质粒,构建了表达载体,转化至大肠杆菌ER2566、BL21。结果:经筛选与鉴定,得到阳性重组子诱导表达后均获得Jo-1融合蛋白。Western blot显示,这两种融合蛋白都具有Jo-1抗原特异性,即仅与含Jo-1抗体的多肌炎和皮肌炎自身免疫病患者血清反应,而与正常人以及188例含有其他自身抗体(分别为RNP阳性、Sm阳性、Ro/La阳性和RNP/Ro阳性)的患者血清均为阴性反应。结论:在两种表达载体中,pMAL-c-Jo-1的表达量超过菌体蛋白的50%,而且以可溶性形式存在,有利于分离纯化,为临床检测创造了条件。     

A new type of perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) in active ulcerative colitis but not in Crohn's disease

Sera of 64 patients with chronic inflammatory bowel disease (IBD) were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) using an indirect immunofluorescence technique on ethanol-fixed human neutrophil granulocytes. 20 of 34 sera (59%) from patients with ulcerative colitis (UC) produced a fine-granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typical diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA, synonym ACPA) seen in active Wegener's granulomatosis (WG). The majority of the 20 p-ANCA positive UC patients had a high inflammatory disease activity. Among the 14 p-ANCA negative UC patients nine were without steroids; five of them had active disease, two were inactive and two had previously undergone colectomy. The remaining five patients still had active disease but received steroids for more than 4 weeks. Only 3 of the 30 sera from patients with Crohn's disease (CD) showed positive p-ANCA reactions. To narrow the specificity of the p-ANCA reaction all 64 sera were tested by ELISA for antibodies against anti-proteinase-3 (WG specific) and on HEp-2 cells for antinuclear (ANA) and anticytoplasmic antibodies. Ten p-ANCA positive UC sera were also tested in a myeloperoxidase ELISA. Only one UC serum reacted positively in the proteinase-3-ELISA and another one produced a weakly positive anti-nucleolar ANA fluorescence on HEp-2 cells. None of the tested sera reacted with myeloperoxidase suggesting that the p-ANCA staining pattern of granulocytes is not restricted to anti-myeloperoxidase antibodies as reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A

BACKGROUND: Ro/SS-A autoantibodies associated with systemic lupus erythematosus (SLE) and Sjögren syndrome may be missed during routine screening for antinuclear autoantibodies (ANA) by immunofluorescence using HEp-2 cells. AIMS: To investigate the use of HEp-2 cells transfected with human 60 kDa Ro/SS-A for routine detection of these antibodies. METHODS: 10,500 sera were screened at a dilution of 1:200 for Ro/SS-A antibodies, identified by intense immunofluorescence staining in 10-15% of hyperexpressing cells of either the nucleus and nucleolus combined or the nucleus alone. RESULTS: Ro/SS-A antibodies were identified in 160/2100 ANA positive sera (8%), of which seven were ANA negative (titre < 200) and 33 had weak ANA titres (200) in 85-90% of non-hyperexpressing "background" cells. Enzyme linked immunosorbent assay (ELISA) confirmed the presence of Ro/SS-A antibodies in 110 newly diagnosed Ro/SS-A positive sera. Of these, 50 reacted with Ro/SS-A, 51 with Ro/SS-A and La/SS-B, and nine with Ro/SS-A and other extractable nuclear antigen (ENA) specificities. Fifteen sera which did not show Ro/SS-A antibodies by immunofluorescence tested positive for Ro/SS-A by immunodiffusion, counter-immunoelectrophesis, or ELISA; of these, 14 had ANA titres > 200. Clinical data from 95 Ro/SS-A positive patients showed that 52% had SLE, 24% Sjögren syndrome, 8% rheumatoid arthritis, and 16% other diseases. CONCLUSIONS: (1) HEp-2 cells transfected with human 60 kDa Ro/SSA are useful for routine immunofluorescence detection for Ro/SS-A antibodies with a positive predictive value of 100%; (2) sera positive for Ro/SS-A antibodies by immunofluorescence should be tested for ENA by other methods because > 50% of these sera will have another ENA reactivity in addition to Ro/SS-A; (3) detection of Ro/SS-A by immunofluorescence may be missed in the presence of high titre ANAs; (4) with a detection sensitivity of 91%, a negative immunofluorescence results for Ro/SS-A does not exclude the presence of this autoantibody.     

Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts

Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated paramagnetic Dynabeads (Dynal) has been used to isolate HSV-2 type-specific gG-2 from solubilized HEp-2 HSV-2 infected cell extracts. The immunomagnetically captured type-specific glycoprotein reacted strongly with monoclonal antibody H1206 and demonstrated a single band with apparent molecular weight of 100000 (100 kDa) and a doublet band with an apparent molecular weight of 60000-64000 (60-64 kDa). We observed the same exact banding pattern when monoclonal H1206 was immunoblotted with Helix pomatia lectin purified HSV-2 gG-2. The immunomagnetically purified gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1 and four human HSV antibody negative sera. In addition, 20 human HSV antibody positive sera obtained from the Centers for Disease Control (CDC), Atlanta, GA, were used for the evaluation of our methodology. Immunoblotting of the human HSV antibody positive samples were in agreement with the CDC HSV serological designation. Sera characterized by reactivity to the immunomagnetically purified gG-2 in conjunction with Western blot has the potential to be used as a confirmatory serological test or to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.     

Molecular cloning of cDNAs expressing SS-B/La protein

Using serum from a patient with Sj?gren's syndrome containing a high titer of anti-SS-B/La antibody, cDNA clones (a representative clone was called pA158) were isolated from a human fibroblast cDNA library in lambda gt11 expression vector. After subcloning of pA158 cDNA into an expression plasmid vector pEX-2, a large amount of the recombinant fusion protein with cro-beta-galactosidase (called pA158EX) was obtained in E. coli culture containing the recombinant pEX-2. Antibodies against pA158EX were purified from the patient serum by Sepharose 4B conjugated with the purified pA158EX protein. Immunofluorescent staining of HEp-2 cells with the anti-pA158EX antibodies showed a speckled nuclear staining. In immunoblot analysis, the anti-pA158EX antibodies reacted with 50 kDa protein that was compatible with SS-B/La protein. Immunoprecipitation of leukocyte lysate with the anti-pA158EX antibodies and the following RNA analysis showed that the antibody precipitated Y5 RNA. These findings indicate pA158 is a cDNA for SS-B/La protein. The purified fusion protein was used for enzyme-linked immunosorbent assay (ELISA). Optical density values of anti-SS-B positive sera were high, but those of anti-SS-B negative sera and healthy donor sera were low. In the Northern blot using human RNA and pA158 cDNA, a single band about 1.8 kb was recognized. A full-length cDNA was further obtained by screening of pcD library using pA158 cDNA as a probe.     

Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis

The presence of autoantibodies in the serum of 110 patients with primary biliary cirrhosis (PBC), 50 with HBsAg negative chronic active hepatitis (HBsAg- CAH) and 30 with HBsAg positive chronic active hepatitis (HBsAg+ CAH) was assessed using two methods: indirect immunofluorescence on cells grown in tissue culture (HEp-2 cell line) or standard mouse tissue sections, and counter immunoelectrophoresis (CIE) with soluble tissue extracts. Anti-nuclear antibodies (ANA) were found in 38% of sera from patients with PBC using HEp-2 cells compared with 10% using mouse tissue. A variety of staining patterns were detected including a pattern of multiple nuclear dots. In contrast, ANA was detected in 70% of sera from patients with HBsAg- CAH and 27% with HBsAg+ CAH. Using CIE four distinct antibody antigen systems were detected: Ro (SS-A), La (SS-B) and two new systems, designated XH and XR, reacting with extracts of human spleen and rabbit thymus, respectively. Correlation of the presence of antibody with clinical conditions confirmed the close association between anti-centromere antibody and sclerodactyly in patients with PBC and indicated an association between 'multiple nuclear dot' staining and the sicca syndrome in PBC. No association was found between the presence of either Ro or La antibody and the sicca syndrome in patients with PBC.     

Comparative evaluation of unfixed and fixed human neutrophils for determination of antineutrophil cytoplasmic antibodies by indirect immunofluorescence.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCAs) are found in the sera of patients with vasculitides and ulcerative colitis. Using indirect immunofluorescence on ethanol fixed neutrophils, ANCAs can be divided into two types: those that give a cytoplasmic staining pattern (C-ANCA) and those that give a perinuclear staining pattern (P-ANCA). Some studies have indicated that the perinuclear staining pattern might be an artefact of alcohol fixation. AIMS: To observe any changes seen in the ANCA staining pattern using indirect immunofluorescence on unfixed neutrophils or neutrophils that had been fixed by ethanol, acetone, or paraformaldehyde. In addition, the effects of the different fixation methods on the sensitivity of the indirect immunofluorescence test were evaluated. METHODS: Twenty one sera from patients with ulcerative colitis and 19 from healthy controls were studied. In addition, 17 sera from patients with vasculitides, including eight with proteinase 3 (PR 3) positive C-ANCA and nine with myeloperoxidase (MPO) positive P-ANCA were included in the study. Antineutrophil cytoplasmic antibodies were analysed by indirect immunofluorescence on unfixed neutrophils or cells fixed by ethanol, acetone, or paraformaldehyde. RESULTS: All the ulcerative colitis associated ANCA positive sera presented a peri-nuclear staining pattern on both unfixed and fixed cells. The acetone and paraformaldehyde fixations decreased the ulcerative colitis P-ANCA titres. Paraformaldehyde fixation also decreased the MPO positive P-ANCA titres. In indirect immunofluorescence, the staining patterns of all the eight PR 3 positive C-ANCA sera and eight of the nine MPO positive P-ANCA sera did not change, even if unfixed or fixed cells were used. CONCLUSIONS: The ANCA staining patterns are not affected by the fixation method used, and are the same as when unfixed neutrophils are used; this suggests that the P-ANCA pattern is not an artefact of alcohol fixation. Furthermore, this study confirms that on ethanol fixed neutrophils, the antigen of ulcerative colitis associated P-ANCA is better exposed than on acetone or paraformaldehyde fixed cells.     

Anti-Golgi Antibody in Rheumatoid Arthritis Patients Recognizes a Novel Antigen of 79 kDa (Doublet) by Western Blot

We have detected cytoplasmic anti-Golgi antibody (AGA) during a routine immunofluorescence test for detecting autoantibodies. Two sera from patients with rheumatoid arthritis (RA) reacted to the Golgi complex by an indirect immunofluorescence technique on HEp-2 cells. Localization of AGA in the Golgi complex was confirmed by double-staining with antibodies to beta-COP. The effect of monensin on the integrity and morphology of the Golgi complex was also studied. To confirm the presence of AGA further, we performed immuno-electron microscopy. Both sera reacted with cytoplasmic antigen located in the Golgi complex of various animal tissues. Furthermore, by using the Western blot technique, both sera reacted to a relative molecular weight (MW) of 79 kDa (doublet) Golgi antigen purified from rat liver. To our knowledge, this study may be the first to identify the relative MW of Golgi antigen by the Western blot method. Identification of this antibody could provide better understanding of protein synthesis and secretion. The presence of AGA in RA patients further substantiates the diversified nature of autoantibody production seen in this disease.     

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。     

Frequency of anti-centromere antibodies in the anti-mitochondrial antibody positive sera

Autoantibodies give us useful informations for the understanding of the co-existence of multiple autoimmune diseases in an individual patient. This study was undertaken to clarify the frequency of anti-centromere antibodies in the sera which were positive for anti-mitochondrial antibodies. Serum samples with anti-mitochondrial antibodies were examined on the frozen rat liver section and HEp-2 cells by indirect immunofluorescence. 57 out of 239 anti-mitochondrial antibody positive sera were found to contain specific autoantibodies reactive with centromeric antigens of the HEp-2 chromosomal spreads. The co-existence of anticentromere antibodies with anti-mitochondrial antibodies were significantly more frequent than with other defined autoantibodies. Clinical observation which shows frequent association of CREST syndrome and primary biliary cirrhosis appear to be explained by the frequent occurrence of anti-centromere antibodies and anti-mitochondrial antibodies.     

Standardization of the immunofluorescence test for autoantibody to nuclear antigens (ANA): use of reference sera of defined antibody specificity

Standardization of the indirect immunofluorescence antinuclear antibody (IF-ANA) test can be improved for a given substrate with use of reference ANA sera, uniform assay conditions, and standardization of optical systems. To accomplish this, reference sera from the Arthritis Foundation with defined antibody specificities for nDNA, SS-B, RNP, Sm, nucleoli, and "speckled pattern" were reacted with commonly used IF-ANA substrates, mouse kidney sections, KB and HEp-2 tissue culture cells. Reagents and assay conditions used were those provided with the substrates in commercially available IF-ANA kits. A microscope slide with graded intensities of fluorescent beads was used to standardize microscope fluorescence intensity readings. The authors' fluorescence pattern and intensity results should be directly comparable to results obtained in other laboratories for the six antibodies for which reference sera are available. Although no defined sera are widely available for SS-A, Scl-70, PM-1, and centromere antibodies, the ability of each substrate to detect these antinuclear antibodies as well as mitochondrial, smooth muscle, ribosomal and microsomal antibodies also was tested. HEp-2 cells and KB cells were found to be superior to mouse kidney sections for detection of SS-A, Scl-70, PM-1 and centromere antinuclear antibodies. Mouse kidney sections were superior for screening of sera for the absence of ANA as well as for detection of smooth muscle and liver-kidney microsomal antibodies. Other antibodies were detected with equal sensitivity with all substrates and each of the three ANA kits used in the study performed satisfactorily. Use of reference sera as well as optical standardization is recommended for IF-ANA testing.     

Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases

The presence of anti-nuclear antibodies (ANA) is a hallmark of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and was recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of serum samples from healthy individuals (HI) have been reported to have a positive ANA test, the majority of which are directed to the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis of SARD, the reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test, significantly reduces the specificity and the positive likelihood of the ANA test. This has significant implications for diagnostic algorithms involving the detection of ANA. We summarize the current knowledge of anti-DFS70 antibodies and their impact on ANA testing. We also suggest a test algorithm which considers the DFS pattern and the presence of anti-DFS70 antibodies. In addition, we describe a novel method based on immunoadsorption of anti-DFS70 antibodies, which increases the specificity of the ANA HEp-2 test for SARD and which has the potential to overcome a significant limitation of the ANA HEp-2 assay.     

Determination of anti-neutrophil cytoplasm antibodies (ANCA) specificity by immunofluorescence on chronic myelocytic leukemia cells.

ANCA positive sera, detected by the standard immunofluorescence method, derived from 37 patients with vasculitis were studied using formalin-acetone fixed chronic myelocytic leukemia cells (CML). All 37 sera were positive on CML cell smears. Furthermore formalin-actone fixation selectively impaired antinuclear antibody binding without reducing ANCA staining and thus facilitated differentiation of these autoantibodies which is often difficult with the standard immunofluorescence method. Two unequivocal and mutually exclusive ANCA binding patterns were identified using the CML smears: (1) type I with diffuse granular binding confined to the polymorphonuclear (PMN) cell lineage and preferentially staining immature cells; (2) type II with similar binding to the PMN cell lineage and, in addition, granular staining of the basophils. All type I antibodies were associated with a c-ANCA pattern suggesting that the major antigen recognized by these antibodies, recently identified as proteinase 3, is not detectable in basophils. The type II pattern was detected in both p-ANCA (84%) and c-ANCA (16%) positive sera. The type I sera remained positive on PMN cells from a myeloperoxidase (MPO) deficient subject and anti-MPO antibodies could not be detected in this group by ELISA. Conversely the type II pattern occurred in the presence of anti-MPO antibodies identified by immunofluorescence, ELISA and dot-blot with the exception of a single serum with antilactoferrin antibody. Type I binding only was observed in Wegener's granulomatosis (WG) but both patterns were found in microscopic polyarteritis (MPA) and rapidly progressive glomerulonephritis (RPGN).     

Identification of secretory immunoglobulin A antibody targets from human milk in cultured cells infected with enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.     

Measurement of antinuclear antibodies: assessment of different test systems

The performance of rat liver and HEp-2 in the detection of antinuclear antibodies (ANA) was studied by two independent sites and compared against an ANA enzyme immunoassay (EIA) screen and EIA systems for the measurement of antibodies to double-stranded DNA (dsDNA) and ENA. Sixty-two sera from patients with connective tissue disease (CTD) and 398 from controls suffering from other disorders were included. The level of agreement was, for HEp-2 and rat liver (within one site), 82.0% (ANA positive/ANA negative) and 51.0% (ANA pattern); and for HEp2- and HEp-2 (between sites), 71.8 and 86.5%. On sera with the ANA homogeneous pattern, the measurement of anti-ENA EIA added little to the detection rate with anti-dsDNA EIA alone. On ANA speckled sera, the EIA reactivity depended on the reaction of the mitotic cells: while sera with positive mitoses reacted similarly to ANA homogeneous sera, in those with negative mitoses the measurement of anti-ENA added about 10% to the detection rate achieved with anti-dsDNA alone. The measurement of anti-Scl-70 and anti-Jo-1 did not markedly improve the positive rate with classical ENA (anti-SSA, -SSB, -Sm, and -RNP) alone, raising doubts about the cost efficiency of including these measurements in unselected sera. The ANA EIA identified patients with CTD at a rate similar to that for rat liver and HEp-2. However, up to 98% of the sera found to be negative by ANA EIA but positive by use of rat liver and HEp-2 were from controls. Thus, the ANA EIA may possible be used as an alternative screen, particularly in laboratories with a high frequency of sera from patients not suffering from CTD.     

Cytoplasmic detection of a novel protein containing a nuclear localization sequence by human autoantibodies

A great diversity of antibodies directed to cell proteins has been described in sera of patients with autoimmune diseases. Most of these sera recognize nuclear components, but some others are directed against cytoplasmic autoantigens. Some of the antibodies directed to cytoplasmic autoantigens are well characterized, such as anti-mitochrondial, anti-ribosomal, anti-microsomal and anti-Golgi complex autoantibodies, but the target of many others remains unknown. In the last 5 years we have selected 32 sera with a characteristic speckled cytoplasmic pattern in indirect immunofluorescence (IIF) assay among a total of more than 31 000 sera from patients with any kind of autoimmune manifestation who attend our Connective Tissue Disease Clinic. Using a human cDNA expression library, we have identified a new autoantibody specificity named RCD-8 in five of these sera, directed to one cytoplasmic autoantigen. Affinity-purified antibodies eluted from a positive clone reproduced the same IIF cytoplasmic staining pattern as native serum and reacted with one single band of 160 kD on an immunoblot of HeLa cell extract. The sequence was found homologous to an autoantigen recently reported named Ge-1, and contains a nuclear localization sequence (NLS), an active protein domain made by a contiguous stretch of amino acids which allows the selective entry of the protein into the nucleus. The five patients whose sera exhibited this new autoantibody specificity displayed different autoimmune pathological profiles.     

Laboratory-based evaluation of "INOVA/QUANTA Lite" to determine antinuclear antibodies (ANA) and autoantibodies to double-stranded DNA, SS-A and SS-B

We evaluated QUANTA Lite reagent series (INOVA Diagnostics, CA, USA) to determine antinuclear antibodies (ANA) and autoantibodies to double-stranded (ds) DNA, SS-A and SS-B, in parallel with MESACUP (Medical & Biological Laboratories, Nagoya). Overall agreements between two reagents for qualitative interpretation ranged from 77.5% (ANA) to 99.0%(anti-SS-B antibodies). When we compared to the results by indirect fluorescent antibody (IFA) test on HEp-2 cells, QUANTA Lite ANA demonstrated better sensitivity and specificity; 92.2% versus 76.5% in sensitivity and 92.1% versus 86.8% in specificity. Also, determining anti-chromatin antibodies and IFA test onto Chrithidia luciliae demonstrated greater interpretive correlation to detect anti-ds DNA by QUANTA Lite than by MESACUP. All the discrepant sera to which QUANTA Lite SS-A gave positive interpretations were confirmed to contain the antibodies specific to SS-A 52kDa antigen, which is supplemented to QUANTA Lite capture-probes. With these results, we can conclude that QUANTA Lite has superiorities over MESACUP; (1) to detect a variety of autoantibodies consisting of ANA, (2) to have a better correlation with confirmatory tests to detect anti-ds DNA antibodies, (3)to detect additional autoantibodies specific to SS-A 52kDa antigen, and (4) to have an enough compatibility in determining anti-SS-B antibodies.