Practical evaluation of methods for detection and specificity of autoantibodies to extractable nuclear antigens

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient's clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.     

Evaluation of multiplexed fluorescent microsphere immunoassay for detection of autoantibodies to nuclear antigens

Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.     

Clinical value of multiplexed bead-based immunoassays for detection of autoantibodies to nuclear antigens

The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes (SS/A, SS/B, Sm, RNP, Scl-70, double-stranded DNA [dsDNA], and centromere B) in both traditional and bead-based assays. The average concordance for the different analytes was 91%, ranging from 81% (dsDNA) to 97% (centromere B). The average relative specificity and sensitivity for the analytes were also high, 92% and 81%, respectively. An examination of 93 "normal controls" demonstrated a 7% false-positive rate, which was comparable to IFA. Percentages of different autoantibodies found in patients with a variety of disease conditions (34 with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; 41 with mixed connective tissue disease; 24 with scleroderma; and 35 with Sjogren's syndrome) were well within the range expected from each group. A scrutiny of results from AtheNA and EIA and Farr results for 185 systemic lupus erythematosus samples revealed comparable results by both methods, with the exception of SS/A and dsDNA, where AtheNA had a higher percentage of SS/A-positive results compared to EIA (51% versus 29%) and a lower percentage of dsDNA-positive results (18% versus 28% at a cutoff of 5 IU/ml).     

Monoclonal autoantibodies to nuclear antigens from murine graft-versus-host disease

Systemic lupus erythematosus-like graft-versus-host (GVH) disease was induced in 10-week-old male (C57BL/10 X DBA/2) F1 mice by the intravenous injection of spleen and thymus cells (2:1) from 10-week-old male DBA/2 mice. GVH mice were bled at regular intervals 1 month after injection. Antibody to nuclear antigens (ANA) were detected by immunofluorescence using HEp-2 cells as substrate, and antibody to histones and DNA were detected by enzyme-linked immunosorbent assay (ELISA). The titer and frequency of ANA were found to relate directly to the number of donor cells injected. In order to determine the spectrum of ANA in GVH disease, mice were reinjected with optimum cell numbers (120 X 10(6], and splenocytes from two mice with high titer ANA were fused to mouse myeloma cell line P3/X63Ag8.653. Hybridomas were analyzed for ANA by immunofluorescence and ELISA. Sixty-eight clones were found which secreted ANA. Of these, 59% produced antibody to double-stranded DNA, single-stranded DNA, and/or histones and the remainder gave a variety of nuclear immunofluorescence patterns including speckled, homogeneous, nuclear matrix, and nucleolar. This study indicates that GVH disease provides an excellent source of splenocytes for the production of ANA-producing hybridomas as well as a model for the study of autoimmunity.     

Identification of specific antibodies to extractable nuclear antigens by passive immunodiffusion.

Extractable nuclear antigens have been identified in a number of systemic rheumatic diseases and may be useful in differentiating those with overlapping clinical features. A number of detection systems have been described but technical feasibility and sensitivity have limited widespread application. We describe a simple immunodiffusion method to identify extractable nuclear antigens with high specificity and sensitivity that compares favorably with parallel hemagglutination studies. This method can be applied easily to routine analysis of antinuclear antigens.     

Recent progress in the study of autoantibodies to nuclear antigens.

Autoantibodies to nuclear antigens can now be classified according to their immunologic specificities. They include antibodies that react with DNA, deoxyribonucleoprotein, nuclear histones, and nuclear acidic protein antigens. It has been established that there are several antinuclear antibodies that react with nuclear acidic proteins, and these include antibodies to Sm antigen, nuclear ribonucleoprotein, and SS-A and SS-B antigens. It has also been established that certain systemic rheumatic diseases, such as systemic lupus erythematosus, Sjögren's syndrome, and scleroderma, are characterized by antibodies of some specificities and not of others. Thus, distinct profiles of antibodies to nuclear antigens may be present, and these may be used as diagnostic aids. Further characterization of these specific nuclear antigen-antibody systems may help in unraveling the etiology and pathogenetic mechanisms of these diseases.     

Significance of extractable nuclear antigens in childhood autoimmune liver disease.

The relative virulence of different isolates of Mycobacterium avium has been linked to their capacity to trigger the secretion of TNF from the macrophages they infect. Smooth opaque (SmOp) variants of Myco. avium have been shown to trigger higher expression of TNF-alpha by macrophages in vitro than the smooth transparent (SmTr) variants. To analyse the role of TNF in resistance to infection by Myco. avium, we studied the infection by two different morphotypes of strain 2.151 of Myco. avium both in vitro and in vivo in the presence or absence of neutralizing antibodies to TNF. No effects were found in vitro regarding the growth of either isolate of Myco. avium. In vivo, only the virulent SmTr morphotype showed enhanced growth in the presence of the neutralizing antibodies. This enhancement occurred relatively late when priming for TNF secretion in vivo was evident. Among four isolates of Myco. avium, three virulent ones induced a marked priming for TNF release and one avirulent strain did not. Mycobacterium tuberculosis H37Ra, which is very active in inducing TNF release due to its lipoarabinomannan moiety, was used to compare with the previous results. The growth of H37Ra in macrophages was increased in vitro by the neutralization of TNF and neutralization of either TNF and/or interferon-gamma (IFN-gamma) enhanced the in vivo proliferation of this microbe in the spleen and liver of infected animals, whereas only the combination of both anti-TNF and anti-IFN-gamma enhanced bacterial proliferation in the lung. We conclude that resistance to the avirulent strains of Myco. avium did not involve TNF, but rather antimicrobial mechanisms expressed constitutively in the mononuclear phagocytes. In contrast, TNF plays an important role in the control of Myco. tuberculosis H37Ra infection.     

Antibodies to extractable nuclear antigens in rheumatoid arthritis: relationship to vasculitis and circulating immune complexes

Antibodies to extractable nuclear antigens (ENA) were analysed in the sera of fifty-two patients with severe rheumatoid arthritis (RA) who were divided into two categories: twenty-five with arthritis only and twenty-seven with extra-articular disease. Using haemagglutination, counterimmunoelectropheresis (CIE) and double diffusion, antibodies to ENA were detected in three (12%) of the patients with arthritis only. In the group with extra-articular disease, antibodies were found in sixteen (59%) of the patients. In ten patients the antibodies reacted with an RNase and DNAse resistant, but trypsin sensitive protein component of ENA. These patients all had extraarticular disease with digital vasculitis being a particularly common feature. Their sera also contained circulating immune complexes detected by elevated cryoprecipitate protein levels associated with relatively low complement levels. It is suggested that antibodies to soluble proteins of nuclear origin may be markers of circulating immune complexes in extra-articular RA.     

The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases

In 1988 and in 1989 consensus workshops were organized in order to define the interlaboratory concordance in detecting autoantibody specificities in selected sera from patients with rheumatoid disorders and to determine the possible causes of discrepancies. In total 20 sera were tested for the presence of antibodies against nRNP, Sm, Ro (SS-A), La (SS-B), Scl-70, centromeric antigens, ribosomal RNP and Jo-1. The methods used for detection by the 28 European laboratories who participated included immunofluorescence, counter-immunoelectrophoresis, immunodiffusion, immunoblotting and ELISA.

The results showed that only a combination of two or more techniques was able to detect all specificities with an adequate efficiency. Recommendations to improve the efficiency of autoantibody detection and to standardize laboratory protocols are given.     

Antibodies against extractable nuclear antigens (ENA) in systemic scleroderma.

Antibodies against nuclear ribonucleoprotein (RNP) were found in 5 of 63 cases of systemic scleroderma, whereas they were present in all but one case of mixed connective tissue disease and in 15 of 67 cases of systemic lupus erythematosus. In all RNP positive cases of systemic scleroderma there were some features of other collagen diseases, and their course was relatively more benign. Studies of RNP antibodies in systemic scleroderma may be of importance for treatment and prognosis.     

Antibodies to extractable nuclear antigens in connective tissue disorders in India: prevalence and clinical correlations

Antibodies to Extractable Nuclear Antigens (ENAs) namely Sm, nRNP, SS-A and SS-B were studied in 397 patients with various connective tissue diseases (CTD), 146 patients with inflammatory polyarthropathies, 16 cases of systemic vasculitides, and 39 normal subjects using counterimmunoelectrophoresis and double immunodiffusion methods. Anti-ENA antibodies were positive in 40.8 percent cases of Systematic lupus erythematosus (SLE) (n = 191), 36.4 percent of overlap CTD (OCTD, n = 44), 27.8 percent of Sjogren's syndrome (n = 18), 10.6 percent of progressive systemic sclerosis (PSS, n = 66) and 2.7 percent of rheumatoid arthritis (n = 111) patients. The correlation of these antibodies with disease features was done. The significant finding was negative association of anti-nRNP antibodies (when present alone) with renal involvement. Anti-Sm antibodies did not correlate with any disease feature. The other associations included correlation of anti-nRNP with pulmonary parenchymal lesions, anti-SS-A with serositis and pulmonary hypertension, and anti-SS-B with myocarditis and recurrent diarrhoea. We conclude that Anti-ENAs may correlate with certain subsets of these diseases but the subject is controversial.     

The measurement of antibodies to extractable nuclear antigen.

By the use of a combination of Ouchterlony immunodiffusion and hemagglutination, it was found that 30 of 81 sera (37%) from patients who had systemic lupus erythematosus (SLE) had antibodies to the ribonucleoprotein component of extractable nuclear antigen, and 37 (46%) had antibody to the Sm antigen. Immunodiffusion was more sensitive for detection of anti-ribonucleoprotein, while hemagglutination detected more anti-Sm. Both technics are necessary to define qualitatively the types of antibodies present in SLE sera.     

Immunodiagnostic value of combined detection of autoantibodies to tumor-associated antigens as biomarkers in pancreatic cancer

Previous studies demonstrated that cancer sera contain antibodies, which react with a unique group of autologous cellular antigens called tumour-associated antigens (TAAs). This study determines whether a panel of TAAs would enhance antibody detection and be a useful approach in pancreatic cancer (PC) detection and diagnosis. The panel of TAAs was composed of six TAAs including p53, p16, p62, survivin, Koc and IMP1 full-length recombinant proteins. Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against these six TAAs in 23 sera from patients with PC and also 23 sera from normal individuals. Antibody frequency to any individual TAA in PC was variable and ranged from 14.7% to 30.4%. With the successive addition of TAAs to a final total of six antigens, there was a stepwise increase in positive antibody reactions reaching a sensitivity of 60.9% and a specificity of 87.0% in PC. Positive and negative likelihood ratio were 4.685 and 0.449, respectively. Positive and negative predictive values were, respectively, 82.4% and 69.0%. Agreement rate and Kappa value were 73.9% and 0.478, respectively. The data from this study support our previous hypothesis that using a panel of appropriately selected TAAs can enhance autoantibody detection in immunodiagnosis of PC. In 15 PC sera with carbohydrate antigen 19-9 (CA19-9) negative, 6 (40%) were found to have anti-TAA (anti-tumour associated antigens) antibodies. When CA19-9 and anti-TAAs were used together as markers in PC detection, the diagnostic sensitivity could be raised from 60.9% to 69.6%. Anti-TAA and CA19-9 were independent markers, and the simultaneous use of these two markers could raise the sensitivity of PC detection.     

Screening test for rheumatic diseases: a combined enzyme immunoassay of rheumatoid factors and antibodies to DNA and extractable nuclear antigens.

Three hundred and one sera from patients with rheumatic and other diseases were investigated using a simple enzyme immunoassay for screening of rheumatoid factors and antinuclear antibodies. The assay had a sensitivity of 77% for systemic lupus erythematosus, 90% for the primary sicca syndrome, and 89% for rheumatoid arthritis. Only 13% of sera from patients with chronic non-rheumatic diseases were positive. The test was further evaluated in a group of patients with suspected rheumatic disease who were followed up for six to 12 months. The test was positive in 16 of 17 sera from patients with connective tissue diseases but in only seven of 36 sera (19%) from patients with non-inflammatory joint diseases. None of the four patients with reactive arthritis was positive by this test. The sensitivity of the assay was comparable with that of the agglutination and immunofluorescence tests for rheumatoid factors and antinuclear factors. For the screening of rheumatoid factor and antinuclear antibodies this kind of test panel offers a simple alternative to the conventional tests for small clinical laboratories and for those in which the autoantibody tests could be automated, as the assay can be performed in one working day and only one dilution of serum is needed to obtain a quantitative result.     

Comparison of tanned cell hemagglutination and counter immunoelectrophoresis test in the detection of antibodies to extractable nuclear antigens (ENA) and to DNA in the systemic lupus erythematosus

A comparative study of tanned cell hemagglutination (TCH) and counterimmunoelectrophoresis (CIE), two easy and reliable methodes for the routine detection of antibodies against nuclear antigens was performed. Antibodies against ENA, RNA-ase sensitive ENA and DNA antigens were searched in patients affected by systemic lupus erythematosus (SLE). Analysis of the results obtained for a particular antibody using TCH and CIE techniques suggests that both methods should be used for the detection of antibodies to nuclear antigens since in several cases antibodies were detected by one method and not for the other. Besides, the frequency of antibodies to ENA, RNA-ase sensitive ENA and DNA revealed by both techniques is similar to the results reported by others employing laborious tests. TCH and CIE serve as a screen to determine the presence of an antibody system and seems to provide the sensitivity enough as to be used in the smaller routine laboratories that wish to provide services for antibodies to nuclear antigens. When tanned cell hemagglutination was used looking for antibodies to DNA or ENA in the sera of patients affected by SLE it proved to be useful since in samples where antibodies to DNA were absent or at very low titres, antibodies to ENA were present in titres ranging from 1:9 to 1:6 561. The authors think this is of considerable importance since the variety of clinical features seen in different subjects with SLE is often accompanied by different specificities or types and amounts of autoantibodies and that certain combinations of these antibodies coincide with specific clinicopathological abnormalities.     

Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. American College of Pathologists

The following guideline presents a series of recommendations based on published medical literature for use of the antinuclear antibody (ANA) test and tests for specific autoantibodies to nuclear antigens in the diagnostic evaluation, prognostic assessment, and monitoring of patients with systemic rheumatic diseases. The guideline emphasizes the need for clinical evaluation to improve the usefulness of test results in patient management. Consideration is given to appropriate use of the generic ANA test in the initial evaluation of patients with signs and symptoms of a systemic rheumatic disease, the evaluation of patients suspected of having lupus erythematosus, use in clinical situations in which the ANA test is required to establish a disease diagnosis, and identification of clinical situations in which the ANA test has little value. Sections are also devoted to recommendations aimed at improving the analytic methods used to detect and measure ANA and specific autoantibodies to nuclear antigens and to the appropriate use of tests for specific autoantibodies in several disease situations that commonly occur in patients with suspected or documented systemic rheumatic diseases. Emphasis is placed on the use of these tests only in situations in which the test results can be expected to provide information necessary for clinical decision making. Those tests of limited medical usefulness and situations in which test results are likely to be misleading are also identified.