In vitro diagnosis of autoimmune diseases
Autoimmune diseases (AID) develop in cases where antibodies (AT) or T cell clones are present in the body, directed against the body’s own antigens (AG). Autoimmune mechanisms are at the heart of many organo-specific and systemic diseases. These diseases include rheumatoid arthritis, insulin-dependent diabetes mellitus, ulcerative colitis and other less known diseases. The resulting autoimmune process is usually chronic and leads to long-term tissue damage. Therefore, the determination of the presence and level of antibodies has a diagnostic and sometimes a prognostic value.
It should be noted that detection of autoantibodies in a particular disease indicates three possibilities:
- Autoantibodies are the cause of the pathological process;
- Autoantibodies are formed as a result of tissue damage caused by a pathological process (for example, in patients with myocardial infarction, the serum may have autoantibodies to cardiac muscle antigens);
- There is some third factor that underlies both tissue damage and antibody damage.
In most cases, however, only the first possibility is realized.
With regard to the diagnosis of AID, three methods are mainly used: immunofluorescence (IF), enzyme-linked immunoassay (ELI) and immunoblotting (IB).
Immunofluorescence can be of two types — direct (DIF) and indirect (IIF). The principle of DIF is that a solution of AT labeled with fluorescent colorant is applied to the cut tissue. AT and AG form complexes, which can then be detected in UV light. The use of IIF technique implies the use of unlabeled AT, the solution of which is applied to the cut tissue, and then reveal the complex AG+AT with the help of fluorochrome-labeled antiimmunoglobulin antibodies (eventually formed a complex AG+AT+antiIgAT).
DIF is used in the diagnosis of such autoimmune disease as systemic lupus erythematosus (SLE). The lupus strip test allows to detect immunoglobulins and complementary factors in dermoepidermal compound of skin biopsies. IIF is used for a variety of rheumatic diseases. For example, the identification of antinuclear antibodies (ANA) is a very reliable diagnostic criterion for a number of autoimmune rheumatic diseases: systemic lupus erythematosus, systemic scleroderma, etc.
Figure 1 ELI Scheme
ELI is a method for detecting antigens using corresponding antibodies conjugated with an enzyme mark (horseradish peroxidase, beta-galactosidase, alkaline phosphatase)
The ELI method consists of 3 main stages:
- Formation of the immune complex “antigen (investigated substance) – specific antibody” or vice versa;
- Formation of the connection of the conjugate with the immune complex formed at the previous stage or with free binding places (determinants);
- Transformation of the substrate under the action of the enzyme label into a recorded signal as a result of biochemical reaction;
ELI is used, for example, in the diagnosis of vitiligo. This method allows to allocate marker autoAT of class IgG with the certain antigenic specificity. In a study conducted in Russia on patients without other comorbidities, it was found that 84% of patients produce autoantibodies against various organ systems. Interestingly, 44% of patients had autoAT to S100 proteins. Such a result can confirm the hypothesis that stress is one of the provoking vitiligo factors.
Immunoblotting is one of the most modern methods, which has become widespread. IB is considered a highly specific and highly sensitive method, so it is used also for patients with uncertain results of tests. Immunoblotting combines several methods of analysis. Initially, the AG is divided by gel electrophoresis. The obtained fractions are transferred to a sheet of nitrocellulose (blot) placed in a special chamber. Then blots are processed with AT for specific AG and radioactively marked conjugate is added to determine the binding of AT+AG. Then the blot is washed and placed in a cassette with X-ray film for the manifestation of complexes. Immunoblotting is used to diagnose autoimmune liver diseases and other pathologies.
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