Epitope mapping
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
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
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).