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Autoimmune Diagnostics

Methods for the Detection of Autoantibodies

In the clinical laboratory, it is important that tests can be carried out rapidly and with as little effort as possible, even when the sample frequency is high. At the same time, the tests must deliver reliable, reproducibly standardized and unequivocal results.

Criteria that determine the quality of a laboratory test include its specificity and sensitivity. These are defined as follows:

  • Diagnostic sensitivity corresponds to the fraction of patients with the disease in question that are correctly identified by the test (the quotient of the number of correct positive test results and the total number of patients with the disease).
  • Diagnostic specificity is the quotient of the number of correctly identified negative test results and the total number of patients/test subjects who do not have the disease in question. This corresponds to the fraction of healthy subjects correctly identified by the test.

Today, the following three methods are preferred for the detection of autoantibodies in the diagnosis of autoimmune diseases:

  1. Immunofluorescence tests on tissue sections or cell cultures (IFT)
  2. Enzyme-linked immunosorbent tests (ELISA)
  3. Immunoblots

For stepwise diagnosis, an immunofluorescence test is often used for initial screening. The result of this test can then be confirmed and differentiated or quantified with the other two test procedures.

The following table gives a comparative overview of these three techniques for the detection of autoantibodies

Tab.1
Antibody detection methods
Detection MethodSubstrateIndicationAdvantagesDisadvantages
Indirect immunfluorescence test (IFT)frozen sections from rat or mouse organs (e.g. liver, heart, stomach)screening for non-organ-specific autoantibodies (e.g. in collagenosis, autoimmune liver diseases)one process allows detection of a whole spectrum of autoantibodies, good sensitivity, very good specificitymuch experience needed for interpretation of fluorescence patterns, leading to high subjectivity
frozen sections of human tissue (e.g. thyroid, pancreas)detection of autoantibodies in organ-specific autoimmune diseases (e.g. thyroiditis, diabetes mellitus I)native antigens in their natural surroundings and conformation are detectedsub-specificities of antibodies cannot be differentiated
cell lines (e.g. HEp-2 cells, Human Epithelial cells)screening, verification of some antibody specificitiesdifferentiation of nuclear antigens possible, also reactions with cytoplasmic antigens can be detectedrelatively frequently detects antibodies with no clinical relevance (high background noise)
Enzyme-linked immunosorbent assay (ELISA)purified antigens, recombinant antigensdetection of individual antibody types as well as defined screeningusually high sensitivity and specificityFalse positive: natural antibodies present in low titres are detected. When using recombinant antigens, reactions with bacterial antigens introduced in production is possible.
used to verify results of IFT and quantificationgenerally high sensitivity and specificity; easy to carry out and standardize, objective measurement technique; titre/activity indicators possible; can be automatedFalse negative: autoantibodies recognize conformational antigens and often react with several determinants of an antigen. Upon deposition on the microtitre plate, antigenic epitopes may be destroyed or altered. Recombinant antigens often do not have the native conformation and do not always represent all determinants.
Immunoblot antigen fractions, purified antigensscreening, simultaneous, rapid detection of various antibodiessimple and fast to carry out test; allows for differentiation of various antigenic determinants, good sensitivity and specificity; estimation of concentrations possible (semiquantitative)False positive: natural autoantibodies can be detected. False negative: antibodies directed against conformational epitopes are sometimes difficult to detect in a blot because they may be changed upon deposition on the membrane.
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