Immunoblot

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An immunoblot ( IB , called immunoblot ) is an immunochemical method. It is used, among other things, in biochemical research and in microbiological and virological diagnostics.

properties

An immunoblot is used to detect an antigen in the course of immunostaining or to demonstrate the specificity of antibodies against defined antigens. In diagnostics, these antigens are predominantly proteins from various viruses or bacteria. The immunoblot is often used as a confirmatory test in diagnostics if an antibody test (e.g. ELISA ) was reactive with the test material and the test result could be false positive . The immunoblot belongs to the immunassay-Procedure. Among other things, an immunoblot can be set up separately for the detection of specific IgG, IgM and IgA. In addition to the specificity test, the immunoblot can also be used to detect neutralizing antibodies against specific pathogen epitopes (for example after vaccination).

The basic principle of the immunoblot is the spatially separate fixation of defined antigens ( blotting ) on an easy-to-use carrier matrix made of plastic or glass fiber, the application of a sample and the detection of the bound, specific antibodies for the corresponding antigen. The antigens are usually applied separately according to size. The oldest form of the immunoblot is the Western blot , in which the antigens are previously separated electrophoretically and then transferred to a membrane. This form of immunoblot was the predominant form until about 20 years ago, but it turned out to be very complex and time-consuming in laboratory diagnostics (usually several days for sample preparation, electrophoresis, blotting and detection of antibodies). Furthermore, dot blots and slot blots performed.

In practice, immunoblots have become established in which defined amounts of antigen are applied to a plastic strip using industrial printing processes, which can be used directly in the laboratory for antibody detection. This type of immunoblot is also known as a line blot . The advantage of the line blot is, in addition to the detection of a specific antibody binding, also the control of the work steps by applied control antigens and the possible detection of a reaction to typically non-specific binding proteins. Antigens from very different pathogens can also be found on the line blot (multiplex method), which enables simultaneous detection.

Another form of immunoblot is used in rapid test procedures (point-of-care testing, POCT), whereby the antigens are fixed on an absorbent matrix and the sample (at the same time the detection reagents for antibody binding) is moved past the fixed antigen by capillary forces ( Immunochromatography ICT, Lateral Flow Test LFT or Lateral Flow Assay LFA).

literature

  • B. Neumeister, HK Geiss, RW Braun, P. Kimmig (eds.): Microbiological diagnostics . 2nd Edition. Stuttgart 2009, ISBN 978-3-13-743602-7 , p. 9
  • D. Raoult and GA Dasch: Line blot and western blot immunoassays for diagnosis of Mediterranean spotted fever . J. Clin. Microbiol. (1989) 27 (9): pp. 2073-2079 PMID 2506223