I am reading a paper in which the researchers use double p:MHCII to stain T cells, but labelled with different fluorochromes to maximise TCR specificity of the assay. Why does this maximise TCR specificity?
Like many other reagents, tetramers can have some non-specific or cross reactive binding. so when you stain with a tetramer and get, say 2% positive cells, you don't know how many of those actually have the correct TCR to bind that tetramer, and how many have some kind of other molecule or structure that binds that particular tetramer. so it's a good idea to stain with a tetramer carrying a different peptide. if a cell binds to both the "correct" and "incorrect" tetramer, you can conclude with some reasonable certainty (though never 100%) that it is not binding via the TCR..
this is an strategy aimed to exclude false positives due to unspecific staining. For example anti-PE responses. Thus only double positives PE/APC (for example) will be truly Tet+. This will be also due with the tetramer control with irrelevant peptide.
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