Method For Detection Of Autoantibodies Research Articles

Evaluating Autologous Serum Skin Test for Early Detection of Auto-Antibodies in Chronic Urticaria

Chronic urticaria (CU) is a condition characterized by recurrent hives and itching lasting longer than six weeks, often with an unclear etiology. Autoimmune mechanisms have been implicated in a significant proportion of CU cases, yet reliable methods for identifying auto-antibodies are still evolving. This study evaluates the effectiveness of the autologous serum skin test (ASST) as a preliminary tool for detecting auto-antibodies in patients with chronic urticaria. The ASST involves intradermal injection of the patient's own serum, with a positive reaction indicating the presence of auto-antibodies that may contribute to the disease. In this study, we recruited 50 patients diagnosed with chronic urticaria, administering the ASST and analyzing the correlation between test results and clinical features. The test's sensitivity, specificity, and predictive values were calculated, and results were compared with other diagnostic methods, including indirect immunofluorescence assays for specific auto-antibodies. Our findings demonstrate that the ASST is a valuable initial screening tool, showing significant concordance with other diagnostic methods and correlating well with clinical severity. The study concludes that the ASST is a practical and cost-effective method for early detection of auto-antibodies in chronic urticaria. Its implementation can guide further diagnostic evaluation and inform therapeutic strategies, offering a more streamlined approach to managing this complex condition. Future research should focus on validating the ASST's role in larger, diverse populations and exploring its potential for routine clinical use.

Predictive autoimmunity using autoantibodies: screening for anti-nuclear antibodies.

Abstract Background: Early detection of antinuclear antibodies (ANA) in asymptomatic subjects is useful to predict autoimmune diseases years before diagnosis. ANA have been determined by indirect immunofluorescence (IIF) using human epithelial type 2 (HEp-2) cells, which is considered the gold standard technique. Multiplex technology (BioPlex ANA Screen) has been introduced for ANA evaluation in recent years. Nevertheless, concordance between BioPlex and IIF is low and there is no harmonization between both methods for detection of autoantibodies. This study has aimed to clarify the clinical significance of autoantibodies detected by BioPlex ANA Screen in subjects with undiagnosed clinical suspicion of autoimmune disease and to determine the predictive value of autoantibodies detected by BioPlex ANA Screen. Methods: A 3-year follow-up study was performed of 411 subjects without a clear diagnosis of autoimmune diseases in whom autoantibodies were detected by BioPlex ANA Screen that were negative by IIF on HEp-2 cells. Results: At 3 years of follow-up, 312 (76%) subjects were positive for autoantibodies by IIF and 99 subjects continued to be negative. A diagnosis of autoimmune disease was found in most of the subjects (87%). Conclusions: BioPlex ANA Screen has greater sensitivity than IIF on HEp-2 cells for autoantibodies detection. Early detection of these antibodies by BioPlex can predict possible development of autoimmune diseases.

Advances in understanding autoimmune pituitary disease: standardized methods for autoantibody detection.

An increasing focus on autoimmune disease has centered on the most commonly affected organ systems. Most of these diseases are associated with a disrupted architecture and function. They result from the infiltration of immunocompetent cells such as T lymphocytes, activation of residential cells, and tissue remodeling (1). In practice, assessing cellular reactivity requires the invasive interrogation of frequently inaccessible tissues. Many diseases in the spectrum of autoimmunity are also associated with production of circulating autoantibodies. In no disease has their importance in clinical decision making been more apparent than in type I diabetes mellitus (2). Despite their frequent lack of disease specificity, several autoantibodies have become invaluable diagnostic markers, and their levels are now routinely assessed by commercial laboratories. They have also been used to judge whether an individual might be at increased risk for developing disease in the future. Therefore, their presence can allow stratification in those who might benefit from increased clinical surveillance. Monitoring many autoantibodies has become a mainstay of managing autoimmune diseases such as those affecting the thyroid, pancreas, and adrenal glands. Furthermore, they have the potential for use as biomarkers for assessing the responses to therapy.

Evaluation of recombinant antigen-based assays for diagnosis of bullous autoimmune diseases.

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.

Computer-assisted classification of HEp-2 immunofluorescence patterns in autoimmune diagnostics

Indirect immunofluorescence with HEp-2 cells presents the major screening method for detection of autoantibodies in systemic autoimmune diseases. Hereby, a large variety of autoantibody entities can be detected and recognized by at least partially typic fluorescence patterns. Currently, this method requires highly specialized technicians and resists automatization. Nevertheless, requirements of good laboratory practice, especially standardization and documentation are hampered by the common microscopic technique. Here, we present a computer-assisted system for classification of interphase HEp-2 immunofluorescence patterns in autoimmune diagnostics. Designed as an assisting system, representative patterns are acquired by an operator with a digital microscope camera and transferred to a personal computer. By use of a novel software package based on image analysis, feature extraction and machine learning algorithms, relevant characteristics describing patterns could be found out. Our results show that identification of positive fluorescence and pre-differentiation between most important HEp-2 staining patterns can be performed by this system. Results and documentation of fluorescence patterns can be integrated into the laboratory system. To enable the usage of such a system in routine diagnostics, accuracy of this system and correct recognition of interferring patterns must be further improved.

Antibodies to sperm basic nuclear proteins detected in infertile patients by dot-immunobinding assay and by enzyme-linked immunosorbent assay.

The auto-antibody response in infertile men was investigated by means of immunoenzymatic methods, dot-immunobinding assay (DIBA), and ELISA, using, as antigens, human sperm basic nuclear proteins. Comparison was made, for the same patients, with antibody response to membrane antigens, detected by tray agglutination test (TAT), spermotoxic test (STT), and immunobead binding test (IBT). A very good agreement was observed between the two kinds of antibody responses. Thus, an ELISA or a dot-immunobinding test with sperm nuclear proteins may be considered as a simple and sensitive method for detection of auto-antibodies in infertile men. The reactivity in ELISA of various synthetic peptides corresponding to sequences of human protamines HP1 and HP2 was also studied: all the sera containing anti-nuclear antibodies do not react with synthetic peptides. This observation suggests that antibodies to sperm nuclear proteins recognize conformational epitopes that are not present on small synthetic peptides.

A Method for Specific Detection of Autoantibodies to the Zona Pellucida in Infertile Women

Human antibodies to porcine erythrocytes were found to bind to porcine but not to human zonae pellucidae, whereas human isohemagglutinins bound to human but not to porcine zonae. On the basis of the binding behavior of agglutinins, it was found that the immunofluorescence of porcine zonae produced by selected sera from infertile women was due to an antibody of different specifity from that which agglutinated pig red blood cells. However, no serum component other than heteroagglutinin was contained in selected sera from control subjects which fluoresced porcine zonae. Since the component was present in the immunoglobulin G fraction, bound to human zonae, and was reactive with zona-specific antigen(s), it was judged an autoantibody to zonae. Thus a method for specific detection of autoantibodies to zonae has been developed by indirect membrane immunofluorescence using porcine ova as targets.

Specificity of auto-antibodies in hemolytic anemia.

The facts that an individual may produce antibodies against his own red blood cells and that such hemolyzing and agglutinating antibodies may be responsible for hemolytic anemia have been known since the beginning of the century. Donath and Landsteiner described in 1904 hemolysins for the patient's own red cells in paroxysmal cold hemoglobinuria. Soon afterward, Widal, Abrami and Brul6,' Chauffard and Troisier, and Chauffard and Vincent incriminated so-called auto-antibodies for some hemolytic anemias. A review of the literature during 30 years following the significant observations of the French investigators reveals only occasional reports on the role of auto-antibodies in hemolytic anemia. The difficulty in demonstrating these antibodies seems to have been the reason. The subject remained dormant until Dameshek and Schwartz revived it by their ingenious experimental studies. They produced hemolytic anemia in guinea pigs with a rabbit immune serum containing hemolytic antibodies against guinea-pig red cells. From these results and from clinical observations, Dameshek and Schwartz postulated the responsibility of an antigen-antibody reaction for certain forms of hemolytic anemia in man. The next important event was the discovery in the middle forties by workers in the Rh field of so-called incomplete antibodies capable of reacting demonstrably only in a favorable environment, provided by a suitable diluent and temperature of incubation. Clumping of red cells becomes manifest in the presence of large molecular diluents (albumin, serum, certain synthetic compounds such as dextran and polyvinylpyrrolidone), but not when one employs physiologic solution of sodium chloride, the time-honored reagent for dilution of antibody containing serum and for suspension of red blood cells. Most significant for the study of auto-antibodies in hemolytic anemia was the introduction by Coombs of the antiglobulin test, the principle of which was discovered by Friedberger and Moreschi.' Another step forward was the demonstration of increased sensitiveness of test red blood cells treated with proteolytic enzymes (trypsin, papain, etc.). The various methods for detection of auto-antibodies are listed in Table 1.