Autoimmune Diagnostics Research Articles

Serum Autoantibodies: From Identification to Clinical Relevance

Serum Autoantibodies: From Identification to Clinical Relevance

Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory.

Indirect immunofluorescence (IIF) plays an important role in immunological and immunometric assays for detecting and measuring autoantibodies. This technology was the first multiplex method used to detect cardinal autoantibodies for the diagnosis of autoimmune diseases. Over the last 20 years, research has enabled the progressive identification of cell and tissue autoantigens which are the target of autoantibodies originally detected by IIF. Accordingly, newer immunometric methods, capable of measuring concentrations of specific autoantibodies directed against these autoantigens, allowed for a gradual replacement of the IIF method in the autoimmunology laboratory. Currently, IIF remains the method of choice only in selected fields of autoimmune diagnostics. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of ANA, the biomedical industry has developed technological solutions which significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. This review summarizes the general and specific features of new available commercial systems (Aklides, Medipan; Nova View, Inova; Zenit G Sight, A. Menarini Diagnostics; Europattern, Euroimmun; Helios, Aesku.Diagnostics; Image Navigator, Immuno Concepts; Cytospot, Autoimmun Diagnostika) for automation of the IIF method. The expected advantages of automated IIF are the reduction in frequency of false negative and false positive results, the reduction of intra- and inter-laboratory variability, the improvement of correlation of staining patterns with corresponding autoantibody reactivities, and higher throughput in the laboratory workflow.

Auto-Antibody Signatures for Disease Diagnosis in Cancer and Autoimmune Diseases

We present examples of autoantibody-signatures from autoimmune diseases and several cancer indications. The results are the output from an integrated platform technology to systematically measure auto-antibody signatures in a vast number of blood samples of patients. It remains a challenge to identify serum markers for disease diagnosis and progression as most markers have limited stability and occur in patient samples in minute and highly variable concentrations. Immunoglobulines (IgG) present as functionally robust class of biomolecules. They can bind human proteins to form autoantibody/autoantigen interaction pairs and are well established kits on the market for autoimmune disease diagnostics. In our UNIarray® technology platform large sets of recombinant human proteins are selected from more than 40,000 recombinant human proteins derived from a fetal brain library. We generate various protein microarrays which contain up to 3200 carefully selected proteins. The proteins are spotted as microarrays and serve as binding partners for any IgG which was formed during development of disease or in response to treatment. One of our lead programs is Prostate Cancer, the most common cause of lethal cancer in men. For improved differentiation between benign prostate disease and malign prostate cancer, we are developing a protein biochip for a specific autoantibody signature. A subset of the marker candidates on the biochip prototype already discriminates prostate cancer patients from persons with elevated PSA and biopsy negative with specificity of 73% and sensitivity of 68%. A verification study of the prostate cancer marker candidates in a multi-centre study worldwide is ongoing. Auto-Antibody Signatures for Disease Diagnosis in Cancer and Autoimmune Diseases Stefan Mullner Protagen AG, Germany doi:10.4172/2155-9929.1000006

Diagnostik der Autoimmunerkrankungen

Autoantibodies play a key role in diagnostic laboratories as markers of autoimmune diseases. In addition to their role as markers they mediate diverse effects in vivo. Autoantibodies with protective effect have been described. Natural protective IgM autoantibodies against tumour-antigens of malignant cells or their precursors may contribute to increased survival rates of carcinoma patients. In a mouse model of systemic lupus erythematosus it has been shown that anti-dsDNA IgM autoantibodies protect from glomerular damage. In contrast, a direct pathogenic role of autoantibodies has been well established e.g. in myasthenia gravis or in Goodpasture syndrome. Similarly autoantibodies against SSA Ro52 are detrimental in neonatal lupus erythematosus with congenital heart block. Moreover, putatively protective autoantibodies may become pathogenic during the course of the disease such as the onconeuronal autoantibodies whose pathogenicity depends on their compartmentalisation. In patients with paraneoplastic syndromes tumour cells express proteins that are also naturally present in the brain. Anti-tumour autoantibodies which temporarily suppress tumour growth can provoke an autoimmune attack on neurons once having crossed the blood-brain barrier and cause specific neurological symptoms. Only a restricted number of autoantibodies are useful follow-up markers for the effectiveness of treatment in autoimmune diseases. Certain autoantibodies hold prognostic value and appear years or even decades before the diagnosis of disease such as the antimitochondrial antibodies in primary biliary cirrhosis or anti-citrullinated protein (CCP)-antibodies in rheumatoid arthritis. It is crucial to know whether the autoantibodies in question recognise linear or conformational epitopes in order to choose the appropriate detection methods. Indirect immunofluorescence microscopy remains a very useful tool for confirmation of results of commercially available immunoassays and for detection of special and rare autoantibodies that otherwise often remain undetected. Standardisation of autoimmune diagnostics is still underway and requires joint efforts by laboratories, clinicians and industry.

EASI—The European Autoimmunity Standardisation Initiative

The European Autoimmunity Standardisation Initiative (EASI) was founded 6 years ago with the intention of improving diagnostics in chronic rheumatic disorders by strengthening the collaboration between clinical and laboratory scientists responsible for autoimmune diagnostics at any given level of the health care systems in Europe. Thorough clinical work-up is frequently not the basis for the usage of laboratory tests. Old established test methods may be exchanged for new methods without the differences being communicated. Often, the optimal way of reporting laboratory results to the clinic has not been agreed upon between clinicians and laboratory scientists. An EASI international team consisting of expert rheumatologists/internists/laboratory scientists from Denmark, France, Germany, Israel, Italy, the Netherlands, Spain, and the United Kingdom meets once a year to discuss how interactions between laboratories and rheumatology clinics could be improved in practical terms, how algorithms for cost-effective and rational autoantibody testing could be harmonized, and what an international concept of standardization in this area could look like. In national EASI teams, which were founded in many European countries, clinical and laboratory experts also meet regularly to discuss how European guidelines may lead to practical improvements in their recent country-specific approaches. To enable the exchange of information and experiences, representatives of the different teams of the EASI Network will meet annually in the EASI Forum. All EASI teams will present and discuss the results of their activities with other experts in the field in an EASI Conference, which will be held biannually at the International Congress on Autoimmunity.

Standardization of Autoimmune Diagnostics in Germany

The German Regional Group of EASI was established during the annual Meeting of the German Society of Immunology in Kiel in September 2005. Since this initial informative meeting, an active core group of about a dozen rheumatologists, immunologists, and laboratory specialists has been generating starter projects. In general, these projects do focus on clinically associated diagnostic questions, and do integrate a variety of specialists with profound knowledge in several related subjects. The aims of the German EASI group are to contribute to the definition of standards and to improve patient care. Therefore, the group is establishing guidelines for the diagnosis of autoimmune diseases, to standardize and improve their quality, combining the experience of clinical and laboratory specialists. The diagnostic activities focus currently on systemic lupus erythematosus (SLE) and on rheumatoid arthritis. These activities include laboratory investigations and diagnosis through clinical manifestations. Standardized diagnostics cannot be based solely on vague symptoms and positive laboratory tests. In laboratory diagnostics, standardization and implementation of objective methods for the detection of autoantibodies has been identified as a central challenge. Here, immune fluorescence techniques and the evaluation of RibP are used as first parameters that could improve SLE diagnostics. Furthermore, guidelines and proposals from scientific medical organizations, and in particular from other national EASI groups will be adapted to the German health system. A cornerstone of implementation is the identification and logistic preparation of existing serum banks, the definition of gaps that should be bridged, and, particularly, the definition and collection of adequate control groups. Through these measures, the German EASI group will provide a standardized diagnostic model of autoimmune disorders throughout Europe starting in the field of rheumatology. Diagnostics may become more rational, efficient, faster, and cost-efficient. Patients with autoimmune rheumatic disorders will profit from receiving an earlier and more accurate diagnosis, which again will allow earlier therapeutic intervention and lead to a better long-term clinical outcome.

Autoimmune diagnostics in diabetes mellitus

Type 1 diabetes results from a specific destruction of the insulin-producing beta-cells of the pancreas. The disease is characterized by the appearance of specific autoantibodies against islet cell antigens. Autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase IA-2 and cytoplasmic islet cell antibodies are useful markers for the differential diagnosis of type 1 diabetes when clinical and metabolic criteria alone do not allow definite classification. Autoimmune diagnostics is of particular importance in adults to discriminate between type 1 and type 2 diabetes and to assess the diagnosis of latent autoimmune diabetes in adults.

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.

Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics - technique, benefits, limitations and applications

Autoimmune diseases are relatively frequent disease complexes, affecting approximately five to seven percent of the population. After cardio-vascular and malignant diseases they come third in mortality. As the clinical diagnosis of rheumatic autoimmune diseases is difficult, laboratory tests are helpful in differential diagnosis and for verification of the clinical diagnosis. The most commonly used assay is the determination of ANAs (anti nuclear antibodies) by indirect immunofluorescence (IFA). However, this method lacks reliable standardisation and is very dependable on the qualification of the observer. Enzyme Immunoassays (EIA) and Immunoblotting techniques, on the contrary, attain good standardisation and comparability. However, the latter methods are limited to the presentation of defined autoantibodies only. There is a need to select a suitable strategy for the use of laboratory parameters in order to support the clinical diagnosis more efficiently. A possible strategy is to replace IFA as a first line screening-step by secondgeneration ANA-EIA kits.

Fully Automated Enzyme Immunoassay System for the Determination of Activator-specific Histamine Release from Basophils in Whole Blood

Histamine release from washed leukocytes and/or whole blood has been extensively utilized for in vitro studies of allergy (1)(2)(3)(4)(5) . It has been shown that when the history and skin test of an individual are positive for a given activator, the probability is very high that the in vitro tests will also be positive for histamine release (6) . Throughout the past 10 years, numerous methods have been developed for the determination of histamine release. The commercially available RIA and enzyme immunoassay (EIA) kits for histamine release are not automated and require the investigator to provide the activators or compounds to be used in stimulating histamine release. This study describes a fully automated basophil histamine release (BHR) EIA method for the determination of histamine in whole blood samples after stimulation of histamine release with various cellular activators available from the manufacturer. The assay is performed on the HY·TECTM System, a fully automated laboratory analyzer with existing methods for allergy and autoimmune diagnostics. All of the components, including the allergen extracts (activators), histamine-coated microtiter plates, buffers, and conjugate, are available from Hycor Biomedical Inc. The HY·TEC BHRTM …

Chemically humanized murine monoclonal antibody against a cell nuclear antigen: usefulness in autoimmune diagnostics.

The cellular nuclear antigen SS-B/La is known to be a major antigenic target to an autoantibody in patients with Sjogren's syndrome and systemic lupus erythematosus. It is useful to detect an anti-SS-B/La antibody from patients' sera in a clinical point of view. We purified SS-B/La from rabbit thymus acetone powder by affinity chromatography with a murine anti-SS-B/La monoclonal antibody (1C3-H7). An enzyme-linked immunosorbent assay method, in which SS-B/La was used to coat a plate, was also successfully established. It is difficult to obtain a large volume of patient's serum with high antibody titer and high specificity as a positive control. We investigated whether or not a positive control from human could be replaced by a murine monoclonal antibody to SS-B/La. The 1C3-H7 was conjugated with a human IgG Fc' fragment using N-gamma-maleimidobutyryloxysuccinimide as a cross-linker. The chemically humanized murine monoclonal antibody (1C3-Fc') was recognized by antiserum specific for human IgG Fc fragment. 1C3-Fc' reacted to SS-B/La but not to other antigens. Furthermore, the titration curve of this conjugate ran parallel with those of patients' sera specific for SS-B/La. It is concluded that a chemically humanized murine monoclonal antibody is useful as a positive control in place of a human patient's serum.

Molecular structure of the La and Ro autoantigens and their use in autoimmune diagnostics

Recombinant autoantigens of the La and Ro specificities have been produced and analyzed using immunological and genetic techniques. Human La cDNA and genomic clones were isolated that encode a protein of 46.7 kDa (408 amino acids). An active La gene consisting of 11 exons was localized on chromosome 2 and has an upstream transcriptional regulatory region resembling ‘housekeeping’ genes as well as elements homologous to the H-ras gene promoter which is presumably a ‘regulated’ gene. A long stretch of alpha helix is predicted in the middle of the La molecule and this region contains an immunodominant epitope. Human Ro protein is encoded by a 1.7 kb mRNA and is 60.6 kDa (538 amino acids) in size. A variety of cDNA clones was isolated and characterized, including those from liver, endothelium, brain and placenta. Long regions of predicted alpha helix also reside in the Ro protein but no sequence similarities to La protein have been found. A possible zinc finger motif that is characterestic of other known nucleic acid binding proteins resides near the middle of the Ro protein. Both the La and Ro proteins contain an extended RNA recognition motif about a region of 80 amino acids but they are less similar in this respect than the known snRNA-binding proteins [1].