Ribosomal antibodies detected by immunofluorescence in systemic lupus erythematosus and other collagenoses

A new ribosomal antibody is described in patients with systemic lupus erythematosus which reacts with all tissues from varied species, giving a distinctive immunofluorescence pattern. This can best be distinguished from other known autoantibodies on stomach where it reacts with chief cells and pancreas where exocrine cells are brightly stained. The ribosomal nature of the antigen was demonstrated by absorption of immunofluorescence and complement fixation studies using purified subcellular fractions. The antigen was unaffected by ribonuclease and was destroyed by trypsin, suggesting that it is one of the ribosomal proteins, while previously reported antibodies were mainly directed against ribosomal RNA, RNA–protein complexes, or polynucleotides and hybrids. The present ribosomal antibody is uncommon and occurs in less than 1% of SLE patients; its clinical significance is similar to that of ribosomal precipitins as the patients had renal involvement and several have died.     

Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays

Two hundred seventy one serum samples from South Korean patients were tested to detect antibodies against Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) and Ehrlichia chaffeensis (the human monocytic ehrlichiosis agent) by indirect fluorescent-antibody assay (IFA) and the Western blot assay. These sera were collected from patients with symptoms of high fever. The rate of seropositivity for Orientia tsutsugamushi was 50.9% by IFA at the Public Health & Environmental Research Institute and National Institute of Health in South Korea. By IFA, 30 (11.1%) and 39 (14.4%) of the serum samples reacted with A. phagocytophilum and E. chaffeensis antigens, respectively. By the Western blot assays, 24 (8.9%) and 29 (10.7%) of the serum samples reacted with purified A. phagocytophilum and E. chaffeensis protein antigens, respectively. This report strengthens other evidence regarding the presence of A. phagocytophilum and E. chaffeensis infections in humans in South Korea.     

Antinuclear Antibodies Detected by Indirect Immunofluorescence on HEp2 Cells and by Immunoblotting in Patients with Systemic Sclerosis

The HEp2 cell cultures appeared highly sensitive in detecting the antinuclear antibodies (ANAb) in systemic sclerosis, principally anticentromere antibodies of the CREST syndrome. The immunoblotting used with either complex cellular extracts from HeLa and rabbit thymus or purified nuclear components (high mobility group (HMG) proteins and histones) is able to identify precisely the ANAb targets and to contribute to diagnosis. With nuclear extracts of HeLa cells, the sera from 75.8% of CREST syndrome subjects stained 18 and 22 kD proteins. Corresponding antibodies were also detected in 72.7% of these patients, on HEp2 centromeres by indirect immunofluorescence. With the same extracts, 33.3% of sera from diffuse sclerosis/acrosclerosis patients contain antibodies staining 86, 73, 32 and 30kD. These sera also stain 77, 66 and 63kD from thymus extracts. Corresponding antibodies will be the anti-SCL-70 antibodies defined by double immunodiffusion. The anti-HMG antibodies were infrequent in systemic sclerosis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and consequently without interest for diagnosis. The anti-whole histones antibodies which are less frequent in diffuse sclerosis/acrosclerosis (35.7%) than in SLE (41.3%) recognize especially HI and H2A in the first diseases, HI and H2B in SLE and HI and H3 in RA.     

Immunoblotting on HEp-2 cells increases the detection of antitopoisomerase 1 antibodies in patients with systemic sclerosis

In order to improve the detection of antitopoisomerase 1 antibodies in a cohort of 111 systemic sclerosis patients, we have performed immunoblots on protein extracts of HEp-2 cells. Using indirect immunofluorescence and ELISA, 27 patients (24.3%) had antitopoisomerase 1 antibodies, 32 (28.8%) had anticentromere antibodies, 31 (27.9%) had antinuclear antibodies with none of these two antibodies and 21 (18.9%) had no antinuclear antibody. IgG from 24/27 (88.9%) patients with antitopoisomerase 1 antibodies bound to 2 protein bands of 63 and 100 kDa identified as topoisomerase 1 by N-terminal sequencing. Antitopoisomerase 1 antibodies were detected in 9 (8.1%) patients who had no antitopoisomerase 1 antibody as determined by ELISA. Patients with antitopoisomerase 1 antibodies had an almost similar phenotype without distinction between ELISA or immunoblot approaches. Our results provide evidence for the use of a combination of ELISA and immunoblot approaches for the detection of antitopoisomerase 1 antibodies.     

Autoantibody detection with indirect immunofluorescence on HEp-2 cells: starting serum dilutions for systemic rheumatic diseases

Antinuclear antibodies (ANA) are determined, among other reasons, to identify samples which need a second test to detect the associated specificities. Our aim was to evaluate the clinical and economic impact generated by using an initial dilution for ANA of 1:160. We analyzed all samples for which ANA, anti-ENA and anti-dsDNA were requested over a 1-year period. ANA were detected by indirect immunofluorescence. Anti-ENA were analyzed with a combination of techniques. Anti-dsDNA were detected by radioimmunoassay. Cost analysis was performed by calculating the difference between two cut-offs (ANA 1:40 and 1:160). A total of 13,233 samples were processed for ANA, of which 59.9% were positive with the 1:40 cut-off and 39.2% with the 1:160 cut-off. At ANA titer 1:40, 0.2% of the samples were anti-ENA-positive and 2.2% were anti-dsDNA positive. Only ANA dilutions of 1:160 and higher showed significantly increased positive predictive value for anti-ENA (1.5 versus 0.2, p=0.029) and anti-dsDNA (8.3 versus 2.2, p<0.001) compared to the 1:40 titer. With the 1:160 cut-off, 16.6% fewer ANA tests, 41.8% fewer anti-ENA determinations and 36.4% fewer anti-dsDNA tests would have been needed. The average saving was 0.87 cost-units per sample (1 unit=2.06euro). We conclude that setting the starting dilution for ANA at 1:160 avoids unnecessary studies, increases the positive predictive values of ANA for anti-ENA and anti-dsDNA, and generates clinical and economic benefits.     

Serology of Mediterranean boutonneuse fever. Kinetics of antibodies detected by 3 methods: indirect immunofluorescence, indirect hemagglutination and latex agglutination

The purpose of this work was to determine the kinetics of antibodies in mediterranean spotted fever as determined by three serologic methods: indirect fluorescent antibody test, latex agglutination test and indirect hemagglutination test. No difference was noticed in the early kinetics but after 6 months, the latex agglutination test and the indirect hemagglutination test did not detect antibodies but the indirect fluorescent antibody test was still positive. This reaction is convenient for seroepidemiologic studies.     

Types of 'reticulin' antibodies detected in human sera by immunofluorescence

Reticulin antibodies have been classified by immunofluorescence into five types reacting with distinct antigens of intra- and extracellular components in mesenchyme. Two types of fibrillar antigens can be distinguished on the basis of the staining patterns, anatomical distribution, and species specificity. A third antibody reacts with either small fibres, amorphous proteins, or mucopolysaccharides lining the hepatic sinusoids (ground substance antigens). In addition, at least two kinds of intrasinusoidal cells show cytoplasmic fluorescence, ie, Kupffer cells and glass-adherent, blood-borne cells antigenically related to peritoneal macrophages. Some sera may contain antibodies reacting with sinusoidal endothelial cells though this has not yet been proven.It has been confirmed that all these distinct antibodies related to reticulin antigens are most frequent in dermatitis herpetiformis and coeliac disease, but they are also found with increased frequency in chronic heroin addicts and in rheumatoid and Sjögren''s syndromes. About 5% of normal individuals had such antibodies and no significant increase could be demonstrated in autoimmune disorders or in liver cirrhosis. The antibodies appear to be stimulated by bacterial or nutritional antigens and are likely to represent anamnestic responses rather than direct cross reactions with a multiplicity of foreign antigens.     

Antinuclear antibodies in patients on anticonvulsant therapy

Antinuclear antibodies to calf thymus nuclei, NP, DNA, sDNA, sNP and Sm antigen were investigated in sera from 170 patients on various programmes of prolonged anticonvulsant treatment. Findings were compared to those on 214 tuberculous patients on isoniazid, 109 SLE patients and 66 healthy subjects.

Patients on anticonvulsants had a significantly higher incidence of ANA to DNA, sDNA, sNP and Sm antigen than the controls but had a lower incidence of ANA to all antigens, except sNP, than the SLE patients. Patients on isoniazid did not have DNA antibodies, but had antibodies to whole nuclei and to NP which were practically absent in the anticonvulsant group.

Of all patients on anticonvulsants only those receiving hydantoins had ANA to Sm antigen, while those receiving only primidone had antibodies to sNP but no antibodies to DNA.

Alteration of sNP with isoniazid did not result in an increased incidence of ANA in the anticonvulsant group as it does in isoniazid treated subjects.

It is concluded that the SLE-activating properties of diverse anticonvulsants probably resides in their potential to induce ANA. Although all anticonvulsants elicit ANA directed primarily to sNP, each may do so by different mechanisms or by altering different sites in the sNP molecule. The mechanisms by which anticonvulsant and isoniazid intake results in ANA probably differ.

Presence of DNA antibodies in some patients on anticonvulsants may indicate that their convulsions were due to SLE.

     

Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay

Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Solid phase assays are increasingly replacing indirect immunofluorescence for detection of antinuclear antibodies. In the most recent generation of solid phase assays, manufacturers attempt to improve the performance of the assays by adding extra antigens.Solid phase assay (EliA CTD Screen, Phadia, in which antibodies to 17 antigens are detected) was compared to indirect immunofluorescence for the detection of antinuclear antibodies in diagnostic samples of 236 patients with autoimmune connective tissue diseases, in 149 healthy blood donors, 139 patients with chronic fatigue syndrome, and 134 diseased controls.The sensitivity of EliA CTD Screen for systemic lupus erythematosus, systemic sclerosis, primary Sjögren's syndrome, mixed connective tissue disease, and inflammatory myopathy was 74%, 72%, 89%, 100%, and 39%, respectively. The reactivity in blood donors, in patients with chronic fatigue syndrome, and in diseased controls was < 4%. Likelihood ratios increased with increasing antibody concentrations. Generally, a positive test result by EliA CTD Screen had a higher likelihood ratio for systemic rheumatic disease than a positive test result by indirect immunofluorescence. A negative test result by indirect immunofluorescence, however, had a lower likelihood ratio than a negative test result by EliA CTD Screen, indicating that the negative predictive value was higher for indirect immunofluorescence than for EliA CTD screen.     

Shifts of anti-Sm-specific antibodies in patients with systemic lupus erythematosus: analysis by counter-immunoelectrophoresis, immunoblotting and RNA-immunoprecipitation.

We investigated serial changes in anti-Sm-specific antibodies by counter-immunoelectrophoresis (CIE), immunoblotting (IB) and RNA-immunoprecipitation (RNA-IP) in three patients with SLE. To assess the immuno-regulatory processes underlying anti-Sm antibody production, we compared changes in anti-Sm-specific antibodies with changes in titres of antinuclear and anti-ds DNA antibodies. IB and RNA-IP detected anti-Sm antibodies earlier than did CIE. The induction of the immune responses against the Sm-specific BB' and D polypeptides occurred separately in two of the three patients. Anti-Sm developed in all three cases after a flare-up of disease, whereas anti-ds DNA antibody levels fluctuated in parallel with disease activity. Thus, anti-Sm and anti-ds DNA antibody production seems to be independently regulated.     

Serotyping of herpes simplex virus isolates: A comparison of BVDU sensitivities,indirect immunofluorescence with monoclonal antibodies,and indirect immunofluorescence with cross-adsorbed rabbit antibodies

Four methods for typing of herpes simplex virus (HSV) isolates were compared for 43 recent clinical isolates and 3 reference strains of HSV, These isolates were subjected to indirect immunofluorescence using both monoclonal antibodies and cross-adsorbed rabbit antisera. Sensitivity to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was also examined.All isolates which fluoresced with HSV-1 monoclonals were found to be sensitive to BVDU with ID₅₀'s ranging from 0.001 to 0.006 μg/ml. All isolates labelled as HSV-2 using monoclonal antibodies had ID₅₀'s to BVDU ranging from 0.4 to 3.5 μg/ml. Results of typing with rabbit cross-adsorbed antisera were less accurate, however. When dilutions were predetermined according to manufacturer's instructions, only 3 of 22 isolates (14%) of HSV-1 were correctly typed. HSV-2 isolates were correctly labelled in 24 of 26 situations (92%).When fluorescence dilution endpoints were compared, however, 21 of 22(95%) HSV-1 isolates had fluorescence endpoints at a greater dilution with HSV-1 antiserum. Twenty-three of 24 HSV-2 isolates were also correctly typed (96%).     

Antinuclear antibodies in Thai patients with connective tissue diseases

A study of antinuclear antibodies (ANA) among Thai patients with various connective tissue diseases revealed that the prevalence of ANA was similar to that in other countries, but that the ANA patterns showed interesting contrasts in most diseases. Rather than the predominant homogeneous pattern seen elsewhere in systemic lupus erythematosus and rheumatoid arthritis, the speckled pattern was commonest among Thai patients with these two diseases (67.9% and 76.9% respectively). Patients with scleroderma exhibited a much lower percentage of the nucleolar pattern (17%) than reported elsewhere. The discrepancy between our findings and those from other studies may reflect differences in genetics, the environment or the severity of disease.     

Antinuclear antibodies and survival in cardiac transplant patients

Serum from 48 patients with cardiac transplants was tested for the presence of antinuclear antibodies (ANA). Thirty-two of the sera were negative for ANA (ANA-), and 16 were positive for ANA (ANA+). Six of the ANA+ sera had a diffuse pattern, six had a peripheral pattern, three had a combined diffuse and rim pattern, and one had a diffuse and nucleolar pattern. Seven of the ANA+ sera were positive at a 1:40 titer, one at a 1:80 titer, four at a 1:160 titer, two at a 1:320 titer, and two at a 1:640 titer. Thirteen of the 32 ANA- patients have died, at intervals of one month to four years after transplantation. Fourteen of the 16 ANA+ patients have died, at intervals ranging from less than one day to four years after transplantation. The mean time period from transplantation to death was 13 +/- 15 months for the ANA+ patients: these were not statistically significantly different. However, the mean interval from transplantation to death of the ANA+ patients with an ANA titer greater than 1:40 was 0.8 +/- 0.8 months, which was significantly different from the mean survival period of the ANA+ group as a whole (P less than or equal to 0.05) and the mean survival of the ANA- group (P less than or equal to 0.005). Although the mechanism is not clear, there appears to be an association between higher titer serum ANA positivity and increased mortality in patients with cardiac transplants with significantly decreased duration of survival after transplantation.     

Comparison of human tumor cells (Hep 2) and rat liver for the detection of antinuclear antibodies by indirect immunofluorescence

Indirect immunofluorescent detection of anti-nuclear antibodies was conducted on sections of rat liver and on smears of human Hep 2 tumour cells in the serum of 1017 patients. Overall, the Hep 2 cells gave titres superior by 1 or 2 dilutions. Taking into account this difference, a good agreement was observed between the 2 cellular reagents in 83 per cent of the sera tested. The divergences, which affect almost one half of the positive sera, are largely due to the presence of anti-centromere antibodies, anti-nuclear antibodies giving a patchy "M3" appearance to the Hep 2 cells and, most importantly, anti-nucleolar antibodies. Such differences demonstrate that there are major qualitative or quantitative antigenic differences between the nuclei of rat hepatocytes and those of Hep 2 cells. Although technically rat liver and Hep 2 cells were found to be fairly easy to use and interpret, only a large comparative study of human pathology will be able to determine which is the better reagent for the routine detection of anti-nuclear antibodies.     

Subtyping influenza A virus with monoclonal antibodies and an indirect immunofluorescence assay

The recent association of certain influenza A virus subtypes with clinically relevant phenotypes has led to the increasing importance of subtyping by clinical virology laboratories. To provide clinical laboratories with a definitive immunofluorescence assay for the subtyping of influenza A virus isolates, we generated a panel of monoclonal antibodies (MAbs) against the major circulating influenza A virus subtypes using multiple inactivated H1N1, H3N2, and 2009 H1N1 strains individually as immunogens. Eleven MAbs that target hemagglutinin (HA) of H1N1 and H3N2 subtypes were selected. These MAbs were combined into three subtype-specific reagents, one each for pan-H1 (seasonal and 2009 strains), H3, and 2009 H1, for the subtyping of influenza A virus-positive specimens by indirect immunofluorescence assay (IFA). Each subtype-specific reagent was tested on 21 prototype influenza A virus strains and confirmed to be specific for its intended subtype. In addition, the subtyping reagents did not cross-react with any of 40 other viruses. The clinical performance of the subtyping reagents was evaluated with 75 archived clinical samples collected between 2006 and 2009 using the D(3) Ultra DFA influenza A virus identification reagent (Diagnostic Hybrids, Inc., Athens, OH) and the influenza A virus subtyping reagents by IFA simultaneously. Sixty-four samples grew virus and were subtyped as follows: 30 as H3N2, 9 as seasonal H1N1, and 25 as 2009 H1N1. RT-PCR was used to confirm the influenza A virus subtyping of these samples, and there was 100% agreement with IFA. This subtyping IFA provides clinical laboratories with a cost-effective diagnostic tool for better management of influenza virus infection and surveillance of influenza virus activity.     

Specific antibodies in serum of patients with hydatidosis recognised by immunoblotting.

Hydatid fluids from sheep, goat, pig and man, after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions, revealed at least 15 discrete polypeptide bands of 8-116 Kda. By ELISA, sera from all 20 cases of hydatidosis showed anti-hydatid antibody, but so did 11 (73%) of 15 sera samples from cysticercosis patients, eight (67%) of 12 sera from patients with other parasitic infections (amoebic liver abscess or hymenolepiasis) and one (4%) of 25 sera from healthy controls. Antibody to cysticercus antigen was found in 14 (93%) of 15 sera from cysticercosis patients, 17 (85%) of 20 sera from hydatid patients, six (50%) of 12 sera from patients with other parasitic infections and one (4%) of 25 sera from healthy controls. Sera from 17 (85%) of 20 hydatid patients, 11 (73%) of 15 cysticercosis patients and five (42%) of 12 patients with other parasitic infections had antibodies to both hydatid and cysticercus antigens. Sera from 20 surgically confirmed cases of hydatidosis reacted with 12 polypeptides of 8-116 Kda in Western immunoblot with hydatid antigens. Polypeptides of 16, 24, 38, 45 and 58 Kda were recognised by all hydatidosis sera but also by many sera from patients with other infections. However, polypeptides of 8 and 116 Kda were recognised by all hydatidosis sera but not by any sera from patients with cysticercosis, other parasitic infections or viral hepatitis, or from healthy controls. Thus, recognition of 8- and 116-Kda hydatid antigens by a patient's serum appears to be a specific test confirming a clinical diagnosis in an individual case of hydatidosis.     

Cell populations and membrane attack complex in glomeruli of patients with post-streptococcal glomerulonephritis: identification using monoclonal antibodies by indirect immunofluorescence

Poststreptococcal glomerulonephritis (PSGN) had been thought to arise from renal deposition of immune complexes and as such is analogous to acute serum sickness. Recent studies of acute serum sickness in animals and PSGN in humans, however, have suggested a pathogenetic role for cellular immunity. To enlarge on these observations, cellular components of glomeruli were characterized by indirect immunofluorescence in 11 tissues from individuals with PSGN using monoclonal antibodies. These studies demonstrate infiltration of glomeruli by monocytes, granulocytes, and lymphoid cells. Focal accumulations of T lymphocytes were also observed adjacent to Bowman's capsule. Analysis of glomerular T-cell subpopulations revealed a predominance of cells reactive with OKT4 early and with OKT8 later in the course of disease. Proliferation of parietal and visceral epithelial cells was associated with increased binding of BA-1 and J5, respectively. The presence of the membrane attack complex of complement was demonstrated by glomerular reactivity with a monoclonal antibody (poly-C9 MA) which recognizes a neoantigen present in poly-C9. Fluorescence was present along the glomerular basement membrane early and within the mesangium late in the course of disease, a distribution similar to that observed for C3 and C5. These observations suggest that immune cells as well as terminal components of complement either provoke or mark tissue injury in PSGN.