Epitope mapping

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The epitope mapping (engl. , Epitope Mapping ) describes the determination of epitopes on antigens . These include both MHCI- bound epitopes, which are recognized by the T-cell receptor on cytotoxic T-cells , and MHCII-bound epitopes, which are recognized by CD4 -positive T cells, as well as soluble or cell membrane- bound antigens, whose epitopes are recognized by antibodies from B cells . The epitope mapping serves to identify immunodominant epitopes for potential vaccines , as well as the development of therapeutic and diagnostic preparations.

Determination of discontinuous epitopes

Detection of discontinuous epitopes by antibodies.

Antibodies can recognize both continuous and discontinuous epitopes. Both types of epitopes can be determined by X-ray crystal structure analysis or by nuclear magnetic resonance spectroscopy , more rarely also by a label transfer , a protection assay or an alanine scan .

Determination of continuous epitopes

Detection of continuous epitopes by antibodies.

Continuous epitopes of antibodies can be detected by ELISPOT or by synthetic peptides which have previously been spatially separated on a membrane (e.g. made of PVDF or cellulose ) or have been synthesized on the membrane by Merrifield synthesis . After the addition of the antibody and a few washing steps with a secondary antibody conjugate, antibody-binding epitopes are detected by immunostaining .

T cell epitopes are exclusively continuous epitopes. While MHC-I-presented epitopes are eight to eleven amino acids in length , MHC-II-presented epitopes are 13-17 amino acids long. T-cell epitopes of cytotoxic and helper T-cells can be determined by ELISPOT or by tetramer staining in flow cytometry . In the case of a protein to be characterized, peptides are synthesized in the desired MHC-binding length, which cover the entire sequence of the protein . In order not to miss any epitopes at the edges of the peptides, an overlap of the peptides of five amino acids (for MHC-I) to eight (for MHC-II) to the peptides adjacent in the amino acid sequence is usually used.

In cytotoxic T-cells can also be the release of radioactive 53 chromium from chromium-containing and (per batch) can be measured with an epitope-laden victim cells after the addition of the cytotoxic T cells. As an alternative to chromium, the antigen-laden sacrificial cells can also be fluorescently labeled and their cell viability monitored. The formation of perforin or granzyme B can also be measured.

Depending on the type, T helper cells secrete characteristic cytokines after contact with MHCII-presented antigens , which can be measured by ELISA instead of chromium .

forecast

A limited prediction of MHC binding of peptides is offered by the SYFPEITHI program or the IEDB , among others . IEDB offers a current database of described epitopes. These predictions only mostly apply, so an experimental verification of the epitopes must follow.

literature

  • C. Janeway et al .: Immunobiology . 6th edition ISBN 0-8153-4101-6 . The 5th English edition is available online on the pages of the NCBI Bookshelf (online) .
  • H.-G. Rammen , J. Bachmann, S. Stevanovic: MHC ligands and peptide motifs . Landes Bioscience, Georgetown, Tx 1997. (International distributor - except North America: Springer Verlag GmbH & Co. KG, Tiergartenstr. 17, D-69121 Heidelberg)
  • H. Rammen, J. Bachmann, NP Emmerich, OA Bachor, S. Stevanović: SYFPEITHI: database for MHC ligands and peptide motifs. In: Immunogenetics (1999), Vol. 50 (3-4), pp. 213-9. PMID 10602881 .
  • R. Vita, L. Zarebski, JA Greenbaum, H. Emami, I. Hoof, N. Salimi, R. Damle, A. Sette, B. Peters: The immune epitope database 2.0. In: Nucleic Acids Res. (2010), Vol. 38 (Database issue): D854-62. PMID 19906713 ; PMC 2808938 (free full text).

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