Performance of a multiplex assay compared to enzyme and precipitation methods for anti-ENA testing in systemic lupus and systemic sclerosis.

INTRODUCTION: Testing for autoantibodies to extractable nuclear antigens (ENA) is essential in the investigation of connective tissue disease. Counterimmunoelectrophoresis is an early described testing methodology for antibodies to ENAs, but is labour-intensive, only moderately sensitive, and reliant on high-quality reference sera. Enzyme-linked immunosorbent assay (ELISA) is automatable for relatively high sample throughput, but has issues with false positives. The addressable laser bead immunoassay (ALBIA) is a multiplex technology which can assess several antibody specificities simultaneously on a small serum sample. We report performance of an ALBIA system compared with CIE and ELISA. METHODS: Samples from 100 systemic sclerosis patients attending Royal Free Hospital in 2007 and 99 SLE patients attending St Thomas's Hospital in 2007-2008 were studied. All samples were tested for antibodies to RNP, Sm, Ro, La, Scl-70, Jo-1 by in-house CIE, FIDIS™ ALBIA (BMD, France), and ELISAs (Phadia, Germany). Cohen's kappa coefficient was used to examine agreement of the different assay methods for the same antibody. McNemar's test was used to detect differences between methodologies. RESULTS: One sample was positive for anti-Jo-1 by CIE, & confirmed by ALBIA & ELISA. All 198 remaining samples were anti-Jo-1 negative by all 3 methods. With respect to RNP, Ro, La, Scl-70 antibodies, there was good agreement in assay performance between CIE, ALBIA, and ELISA. For Sm, agreement was less good between CIE and ELISA (kappa 0.491), and ALBIA and ELISA (kappa 0.403). Using McNemar's test performance was no different between the 3 assays, with the following exceptions: between CIE and ELISA for Ro-60 (p<0.01) and RNP (p<0.05), and between ALBIA and ELISA for RNP (p<0.01). CONCLUSIONS: The FIDIS™ ALBIA produced similar level of performance as CIE, but with advantages of automation, and less dependence on highly skilled operators. ALBIA represents a potential advancement applicable to routine Immunology diagnostics.

Thyroid autoimmunity in chronic urticaria.

BACKGROUND: Chronic urticaria (CU) is an autoimmune process in some patients. An association between CU and autoimmune thyroid disease has also previously been proposed. Our group has identified functionally significant histamine-releasing autoantibodies in one subset of CU patients (subset 1), predicted by positive autologous intradermal serum tests and positive histamine release from donor basophil leucocytes in vitro. Sera from a second subset of patients (subset 2), all of whom had positive autologous intradermal serum tests, failed to release histamine from donor basophils. A final disease subset (subset 3) has no identifiable skin reactivity (negative autologous serum skin test) or in vitro histamine releasing activity. OBJECTIVES: In order to examine further the possible relationships between thyroid autoimmunity, thyroid dysfunction and CU, we have examined thyroid autoantibodies and thyroid-stimulating hormone (TSH) levels (an indirect measure of thyroid dysfunction) in the three CU subsets. PATIENTS/METHODS: We studied 182 patients (69% female), of whom 90 had a positive autologous intradermal serum test. RESULTS: Eighteen skin test-positive and four skin test-negative patients had thyroid microsomal antibodies (TMA). TSH outside the normal range was found in 13 skin test-positive and one skin test-negative patient. These findings represent clustering of TMA positivity [risk ratio (RR) 4.06, 95% confidence interval (CI) 1.56-10.6] and of abnormal thyroid function (RR 15.5, CI 2.07-11.6) among the skin test-positive patients. However, in the overall study group an elevated TSH was present in seven patients (3.8%, CI 1.6-7.8) comparable to the 5% expected prevalence in the community. Thyroglobulin antibodies (TGA) were present in two of 182 patients. CONCLUSIONS: There were significant differences between skin test-positive and skin test-negative patients with regard to autoimmune thyroid disease. Evidence for autoimmune thyroid disease and abnormal thyroid function was largely found among the skin test-positive patients, supporting the theory of an autoimmune aetiology in this group.

Antibodies to beta2-glycoprotein I: a potential marker for clinical features of antiphospholipid antibody syndrome in patients with systemic lupus erythematosus.

OBJECTIVE: To clarify risk factors for the development of clinical features of antiphospholipid syndrome (APS) in patients with anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). METHODS: We studied 65 SLE patients, all with positive IgG and/or IgM aCL. Patients were divided into 2 groups; I: 29 SLE patients with features of APS (SLE/APS) and II: 36 aCL positive SLE patients without any feature of APS (SLE/aCL). Serum samples were collected from our serum bank. Anti-beta2-glycoprotein I (anti-beta2-GPI) were tested by ELISA using irradiated plates in the absence of cardiolipin. Anti-dsDNA antibodies were tested by standard Farr assay. RESULTS: There were no major differences between SLE clinical manifestations in both groups. However, the frequency of IgG anti-beta2-GPI was markedly increased in SLE/APS (18/29, 62%) than in SLE/aCL (4/36, 11%) (chi-squared 18.6, p=0.0001). The levels of anti-dsDNA antibodies in the same samples were slightly lower in SLE/APS. CONCLUSION: Our data suggest that increased levels of IgG anti-beta2-GPI may be a specific feature of SLE/APS patients rather than reflecting a polyclonal B cell activation.

Value of immunologic testing in stroke patients. A prospective multicenter study.

BACKGROUND AND PURPOSE: The aims of this prospective and multicenter study were to determine the frequency of anticardiolipin and antinuclear antibodies in an unselected ischemic and hemorrhagic stroke population and to evaluate the clinical significance of these autoantibodies. METHODS: Over a 1-year period, we collected plasma from 481 consecutive patients with ischemic or hemorrhagic stroke attending four different hospitals. Blood (10 mL) was drawn from each subject into a citrated glass tube. Plasma was obtained immediately by centrifugation and was stored at -70 degrees C until use. Concentrations of IgM and IgG anticardiolipin antibodies were measured at room temperature in normal (not heat-treated) plasma by standardized enzyme-linked immunosorbent assay. All sera were treated by indirect immunofluorescence on mouse liver and kidney sections for antinuclear antibodies. RESULTS: A total of 481 patients (325 men, 156 women) 16 to 90 years in age (mean age, 61 years) were studied. Anticardiolipin antibodies were present in 5 of 481 (1.04%) patients. One patient was IgG positive and four patients were IgM positive. Of 481 patients, 35 (7.2%) were positive for antinuclear antibodies. Anti-DNA antibodies were not demonstrable in any patient. CONCLUSIONS: The frequency of anticardiolipin antibodies in a heterogeneous stroke population is possibly lower than reported. The routine screening of anticardiolipin and antinuclear antibodies in a stroke population is of questionable value.

Isotype profile and clinical relevance of anticardiolipin antibodies in Sjögren's syndrome.

The purpose of this study was to determine the occurrence and clinical value of anticardiolipin antibodies in patients with Sjögren's syndrome. Thirty one patients with primary Sjögren's syndrome (all women, mean (SD) age 48.3 (11.2) years) and 32 patients with secondary Sjögren's syndrome with rheumatoid arthritis (all women, mean (SD) age 54.9 (11) years) were studied. IgG, IgM, and IgA anticardiolipin antibodies were determined by a standard enzyme linked immunosorbent assay (ELISA) technique. Anticardiolipin antibodies were found in five patients (16%) with primary Sjögren's syndrome and in seven patients (22%) with secondary Sjögren's syndrome. There was no correlation between anticardiolipin antibodies and the clinical features of the antiphospholipid syndrome (thrombotic events, fetal loss, thrombocytopenia) or extraglandular manifestations of Sjögren's syndrome (arthritis, skin lesions, myositis, polyneuropathy, central nervous system disease, pulmonary and renal disease) in either group. Among the various serological features studied, anticardiolipin antibodies correlated with antinuclear antibodies and antibodies to RNP only in patients with primary Sjögren's syndrome. These results indicate that although anticardiolipin antibodies are often found in serum samples from patients with Sjögren's syndrome, their clinical significance remains unclear.

A prospective evaluation of antithyroid antibody prevalence in 100 patients with systemic lupus erythematosus.

During a 6-month period, 100 patients with systemic lupus erythematosus (SLE) were consecutively studied for the presence of antithyroid antibodies and thyroid disease. Overall, the prevalence of antithyroid antibodies was similar in patients with SLE (21%) and controls (16%). However, antithyroglobulin antibodies were found in 11% of patients with SLE and only 2% of controls (p = 0.009). The levels of antimicrosomal antibodies were also different (median levels: SLE = 400; controls = 100) but this difference did not reach statistical significance. We found a significant correlation between thyroid stimulating hormone (TSH) levels and the levels of both the antithyroglobulin dilutions (p less than 0.05) and the antimicrosomal dilutions (p less than 0.05). This was not seen in the controls. A higher frequency of clinical thyroid disease was seen in patients with SLE with thyroid antibodies (5/21; 3 hypothyroid, 2 hyperthyroid) than in those without these antibodies (1/79; p = 0.001). Levels of antithyroid antibodies correlated with clinical or subclinical (marked by elevations of TSH) thyroid disease. Patients with SLE with these antibodies were significantly older (mean age 47.5 +/- 13 years) than those without antithyroid antibodies (mean age 37.5 +/- 12 years; p less than 0.001). Antithyroid antibodies define a subset of older patients with SLE with increased prevalence of both clinical and subclinical thyroid disease.

Antibodies to endothelial cells in systemic lupus erythematosus: a potential marker for nephritis and vasculitis.

Using an ELISA, anti-endothelial cell antibodies (AECA) have been found in sera obtained at the time of renal biopsy in 46 out of 57 patients (81%) with systemic lupus erythematosus (SLE) and nephritis (mean binding index (BI) = 84% +/- 52.8) compared with 22 out of 50 SLE patients (44%) without nephritis (mean BI = 45% +/- 35.9). Seventy normal human sera had a mean BI of 10% +/- 9.8. The highest levels were seen in patients with diffuse proliferative glomerulonephritis (WHO grade IV) and in patients with proteinuria and nephrotic syndrome. When the biopsies were assessed for activity and chronicity scores, AECA were associated with active renal lesions (P less than 0.001). AECA levels correlated with low complement levels but not with anti-DNA antibodies to extractable nuclear antigens (ENA), anti-cardiolipin or anti-neutrophil cytoplasmic antibodies. The presence of AECA conferred a positive predictive value of 0.68 for the presence of nephritis. Twenty-five patients had active vasculitis at the time of assay and the highest AECA values were seen in patients with both nephritis and vasculitis. No correlation was seen with serum immunoglobulin levels and immune complexes did not bind significantly to the endothelial surface. The possible role of these antibodies as a marker in lupus nephritis is discussed.

Cell lines of novel type derived from a diabetic secrete tissue-reactive human monoclonal antibodies.

Human monoclonal antibodies have been produced from lymphocytes of an acute-onset insulin-dependent diabetic patient. Peripheral blood lymphocytes were hybridized with a fusion partner HMY-1320. Initial screening of human immunoglobulin secretion was made by a nitrocellulose dot blot assay. Ten stable cell lines of novel type, secreting human immunoglobulin, were obtained. These cell lines have been maintained in continuous culture over 6 months and cryopreserved in liquid nitrogen for 14 months. Human monoclonal antibodies of IgG and IgM class have been produced and are secreted at a rate of 150-650 ng/ml/10(6) cells/day. Monoclonal antibodies were tested for histological staining against a variety of endocrine and non-endocrine tissues. One monoclonal antibody, LT1E12, demonstrates a staining pattern in human, rat, and mouse tissues, similar to that of mitochondrial antibodies. Another antibody, LT3C4, demonstrates weak staining of smooth muscle in rat and mouse kidney sections. Neither specificities were detected in the diabetic patient's serum. The variety of immune tissue specificities obtained in this study demonstrates the potential value of human monoclonal antibodies as probes to analyze the complexity of autoimmunity in diabetes mellitus.

Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins.

Sera from 75 patients with monoclonal gammopathies and with no clinical evidence of autoimmune disease have been screened for a wide range of autoreactivity including binding to DNA, cardiolipin, extractable nuclear antigen (ENA), rheumatoid factor activity and the presence of the common anti-DNA antibody idiotype PR4. The sera of 17/75 (23%) patients possessed autoreactivity: six were positive for anti-DNA activity, two had anticardiolipin activity and the PR4 ID was found in two sera (both of which possessed anti-DNA activity). Antibodies to ENA were found in one serum (anti-Ro) and anti-organ-specific antibodies in five. Using iso-electric focusing and immunoblotting we have shown that the PR4 ID and DNA binding activity are carried on the paraprotein and not on some other serum constituent. The IgG subclass distribution of 55 IgG paraproteins has also been investigated. The majority of IgG paraproteins belong to IgG1 subclass (55%), with the others, being IgG2 (4%), IgG3 (9%) and IgG4 (27%). In this study we have shown that sera from myeloma patients frequently possess autoreactivity, and that in many cases this can be attributed to the paraprotein.

A comparison of autoantibodies and common DNA antibody idiotypes in SLE patients and their spouses.

In order to determine whether environmental influence per se might influence autoantibody production, sera from the spouses of 20 SLE patients were examined. No antibodies to cardiolipin, poly (ADP-ribose), or ENA were detected and none had detectable rheumatoid factor. One weakly positive ANA reaction was noted, one had anti-DNA antibodies (by RIA and ELISA) and in two sera the common DNA antibody idiotype 16/6 was found. The idiotype was not, however, present on either anti-DNA or anti-K30 antibodies. Although long-term analyses are required, it is evident that sharing the same environment with patients who commonly express a wide range of autoantibodies and common idiotypes rarely leads to their expression in non-autoimmune subjects.

Profile of autoantibodies in the serum of patients with tuberculosis, klebsiella and other gram-negative infections.

Autoantibody profiles were examined in the sera of untreated patients with tuberculosis, and those with klebsiella septicaemia, and klebsiella and E. coli urinary tract infections. Rheumatoid factors of the IgM, IgA and IgG isotypes, antinuclear antibodies and antibodies to poly(ADP-ribose) were all frequently detected (generally 15-40%). In contrast, antibodies to the extractable nuclear antigens and to the organ specific antigens were unusual (generally less than 10%). In comparison, in a group of lupus patients IgM rheumatoid factor, anti-nuclear antibodies, antibodies to poly(ADP-ribose) and antibodies to the extractable nuclear antigens were more frequently found, but IgA and IgG rheumatoid factors and antibodies to the organ-specific antigens were present in much the same frequency.

Autoantibodies to cartilage and type II collagen in relapsing polychondritis and other rheumatic diseases.

Cartilage antibodies were demonstrated by indirect immunofluorescence (IFL) on human fetal cartilage in 6 out of 9 patients with relapsing polychondritis (RPC), in 4 out of 260 patients with rheumatoid arthritis (RA), and in only 1 out of 1016 patients with other disorders. The antibodies were specific for cartilage and evenly stained the whole cartilage matrix. They were predominantly of IgG class and varied in titres from 1:1 to 1:320. Follow-up studies in the RPC patients indicated that higher titres were present during the early acute phase of the disease. Five of the 6 positive cases had developed the disease within the past 12 months, and the 3 negative cases had had the disease for 3 to 7 years when tested. The RA cases showing positive cartilage IFL had no clinical evidence of RPC. Sequential measurements in 2 of the 4 cases showed that these antibodies became detectable some years after the onset of arthritis. Absorption studies with human type II collagen and purified porcine proteoglycan failed to remove the cartilage IFL. Antibodies to human native type II collagen were measured by an enzyme-linked immunosorbent assay. The highest levels were found in the RA sera which also displayed cartilage IFL, but the 2 tests gave discordant results. RPC sera showed the same antibody levels by this method, as did cartilage-IFL-negative RA sera, though both groups had higher mean levels than health controls. The findings that cartilage antibodies are detected in the majority of cases of RPC and only rarely in other diseases suggests these antibodies may play an important role in the pathogenesis of cartilage destruction in RPC.

An evaluation of two new haemagglutination tests for the rapid diagnosis of autoimmune thyroid diseases.

Two haemagglutination tests using preserved turkey erythrocytes are described for the detection of thyroglobulin and microsomal antibodies, respectively. Comparative studies with the more traditional sheep cell techniques show good correlation of titres when testing sera from patients with autoimmune thyroid disorders.

A human-specific mitochondrial antibody its importance in the identification of organ-specific reactions.

A previously unrecognized autoantibody, detected by immunofluorescence, reacted with all human organs but gave negative results on tissues from rat, mouse, rabbit, guinea-pig, calf and chicken. From its predilection for mitochondria-rich cells (oncocytes) and its selective absorption with human but not animal mitochondria, it was identified as an anti-human mitochondrial antibody and named AHMA. The antibody is found in about 1% of normal subjects and is mostly of IgG class and of low titres. Its prevalence is increased in primary biliary cirrhosis where it may be associated with the standard non-species-specific AMA used for the differential diagnosis of this disease. The importance of AHMA is mainly in possible confusion with organ-specific reactions in submaxillary duct, parathyroid oxyphil cells and in trying to identify new endocrine cells such as those producing pancreatic polypeptide (HPP) in human tissues. Animals immunized with human hormones develop reactions to human mitochondria and thus produce misleading immunofluorescence reactions when used in low dilutions.

Classification of smooth muscle autoantibodies detected by immunofluorescence.

Three hundred and twelve sera containing antibodies to smooth muscle (SMA) wer analysed for the immunofluorescence patterns they produced in various tissues. A classification is described based on the three main appearances in rat kidney. Some sera, mainly of low titre, reacted only with vessel walls (SMA-V), some stained vessels and renal glomeruli (SMA-G) and high titre sera, mainly from patients with chronic active hepatitis stained vessels, glomeruli, and intracellular fibrils in renal tubules (SMA-T). Peripheral staining in hepatocytes or thyroid cells was not a regular feature. 41/43 polyclonal SMA-T and -G sera were absorbed out completely by actin, and this also removed the pericullular staining in liver and thyroid when present. High titre SMA-V antibodies could not be absorbed by actin, and the antigen remains to be identified.