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Methods for the detection of autoantibodies
In the clinical laboratory, it is important that a test can be carried out quickly and with as little effort as possible, even with a large number of samples. At the same time, the tests must deliver standardized and unambiguous results reliably and reproducibly.
Decisive criteria for the quality of a laboratory test are its specificity and its sensitivity. They are defined as follows:
- The diagnostic sensitivity corresponds to the proportion of patients with the respective disease that the test correctly detects. (Quotient of the number of correctly positive test results and the total number of patients with this disease.) This corresponds to the probability that the test will show a positive result if the target marker is present.
- The diagnostic specificity results from the quotient of the number of correctly negative test results and the total number of patients / test persons without the corresponding disease. It corresponds to the proportion of healthy subjects who the test correctly recognizes. This corresponds to the probability that the test will show a negative result in the absence of the target marker.
For the detection of autoantibodies in the diagnosis of autoimmune diseases, the following three methods are preferred today:
- Immunofluorescence tests on tissue sections or on cell cultures (IFT)
- enzyme-linked immune absorption tests (ELISA)
For step-by-step diagnostics, an immunofluorescence test is often carried out first for screening, the result of which is then confirmed and differentiated or quantified by one of the other two methods.
The following table gives a comparative overview of these three methods for the detection of autoantibodies
- Tab. 1
- Methods of autoantibody detection
|Indirect Immunofluorescence Test (IFT)||Frozen sections of organs of the rat or mouse (e.g. liver, kidney, heart, stomach)||Screening for non-organ-specific autoantibodies (e.g. for collagenoses, autoimmune liver diseases)||one approach allows the detection of a whole spectrum of autoantibodies; good sensitivity, very good specificity||a lot of experience is required to interpret the fluorescence pattern, resulting in a high level of subjectivity|
|Frozen sections of human tissue (e.g. thyroid, pancreas)||Detection of autoantibodies in organ-specific autoimmune diseases (e.g. thyroiditis, diabetes mellitusI)||native antigens in their natural environment and conformation are detected||Antibody subspecificities cannot be differentiated|
|Cell lines (e.g. HEp2 cells, Human epithelial cells)||Screening, detection of some antibody specificities possible||Differentiation of core antigens possible, reactions with cytoplasmic antigens can also be detected||detects relatively common naturally occurring antibodies without clinical relevance (high background)|
|Enzyme-linked immune absorption test (ELISA1))||purified antigens, recombinant antigens||Detection of individual antibody specificities is just as possible as a defined screening||mostly high sensitivity and specificity||False positive: low-titer natural autoantibodies present are recorded; in the case of recombinant antigens, reactions with any bacterial antigens still present from production are possible.|
|serves to confirm findings from the IFT and for quantification||easy to implement, easy to standardize, objective measurement method; Titer / activity information possible; can be automated||False negative: autoantibodies recognize conformational antigens and often react with several determinants of an antigen. When attaching to the microtiter plate, antigenic epitopes can be destroyed or changed. Recombinant antigens often do not have the native conformation and do not always represent all determinants.|
|Immunoblot||Antigen fractions, purified antigens||Screening, simultaneous rapid detection of different specificities||Immunoblot can be performed quickly and easily; allows differentiation of different antigen determinants; good sensitivity and specificity; Concentration estimates possible (semiquantitative)||False positive: natural autoantibodies can be detected. False negative: Antibodies directed against conformational epitopes are sometimes difficult to detect in the blot because they are changed when they are applied to the membrane.|