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An MHC tetramer assay or simply tetramer assay or tetramer stain is a procedure developed at Stanford University School of Medicine used to detect and quantify T-cells that are specific for a given antigen within a blood sample. T-cells are part of the cell-mediated immune response and possess one receptor, i.e. T-cell receptor (TCR), and one co-receptor, i.e. either CD4 or CD8. In order for a T-cell to be activated, its CD co-receptor must bind to the appropriate major histocompatibility complex (MHC) on the surface of an antigen presenting cell, while the TCR must bind the peptide being presented on the MHC. T-cells possess variable TCRs that recognize different peptides. These receptors may only bind certain sequences or configurations of peptides from antigens, and so each T-cell is specific for a given antigen (disregarding cross-reactivity), namely T-cell clone. Thus, out of all of the T-cells in a population, only few may be specific for a given peptide. Generally, if a person’s immune system has encountered a pathogen, this individual will possess T-cells with specificity toward some peptide on that pathogen. Hence, if a tetramer stain specific for a pathogenic peptide results in a positive signal, this may indicate that the person’s immune system has encountered and built a response to that pathogen. The tetramer itself consists of multiple bound MHC molecules. The need for an MHC tetramer arises from the high dissociation rate of MHC monomers, making monomers difficult to use as a detection strategy. Tetramers however, can bind multiple MHCs at a time to a T-cell (ideally, 3 of the 4 MHCs would bind) and so increase the binding avidity and circumvent the problem of dissociation. The centerpiece of each tetramer is a streptavidin complex. Streptavidin is a molecule that forms homotetramer complexes, with each monomer having an unusually high affinity for biotin. Exploiting these facts, scientists have bioengineered E. Coli to produce soluble MHC molecules with a biotinylation protein domain, meaning a part of the MHC can be replaced by covalently bound biotin (via BirA enzyme activity). The MHC molecules must then be mixed with the antigenic peptide of interest, forming peptide-MHC (pMHC) complexes. The biotinylated domain then allows for up to 4 pMHCs to bind to a fluorescently tagged streptavidin complex with high affinity. The resulting pMHC-streptavidin-fluorophore tetramer can be added to a sample of cells. The tetramers bind to T-cells that are specific for both the MHC type and peptide being used in the tetramer. Once the tetramers are bound, T-cells are often stained with other fluorophores and the sample is washed to remove non-bound tetramers and ligands. The stained sample is then run through a flow cytometer for detection and sorting. The fluorophore on any bound tetramers can be excited to give a signal, indicating that the tetramer is bound to a T-cell, and thus that the bound T-cell is specific for the peptide antigen of interest. Ultimately, a signal means that there exists some cell-mediated immune response to the pathogen from which the antigenic peptide is derived, and the strength of the signal gives the strength of the immune response. ==References== * 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Tetramer assay」の詳細全文を読む スポンサード リンク
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