KatieJane Posted January 6, 2009 Share Posted January 6, 2009 My colleague and I are confused as to whether you can detect a receptor expressed on a cell surface to the same degree when it's bound by its ligand. For example, when dendritic cells secrete the cytokine IL-12, expression of its receptor is up-regulated. In trying to quantify this receptor expression by antibody detection/flow cytometry, we are worried that the binding of the IL-12 ligand may jeopardise the quantification of the receptor. Does it depend of the respective binding sites of the antibody and ligand? Does ligand binding always affect the capacity of the antibody to bind? Can anybody help?! Link to comment Share on other sites More sharing options...
ecoli Posted January 6, 2009 Share Posted January 6, 2009 To clarify... you have an antibody that will bind the receptor, but if the ligand binds, this could block your antibody from binding to the receptor? Link to comment Share on other sites More sharing options...
KatieJane Posted January 6, 2009 Author Share Posted January 6, 2009 yes, that's right, could the ligand block binding of the antibody? Link to comment Share on other sites More sharing options...
CharonY Posted January 6, 2009 Share Posted January 6, 2009 It depends what is recognized as the epitope. Even then it depends on the amount of conformational changes upon binding to it. If you want to make a very precise quantitation, one should use (monoklonal) antibodies directed against an epitope not undergoing conformational changes upon ligand binding, of course. If your goal is just to see whether there is am up-regulation, it may mot be an issue, as with more receptors you would also see more signal. There might be a competition with the ligands, however, so this would only be a qualitative analysis. But again, it really depends where the epitope is. If it is not in the pocket it should work reasonably well (depending on the precision you require). Link to comment Share on other sites More sharing options...
GDG Posted February 17, 2009 Share Posted February 17, 2009 If you're using a commercial MAb, the supplier's literature should tell you whether or not the MAb competes for binding with the ligand. If you're using home-grown MAbs, you'll probably need to test directly to see if they compete for binding with IL-12. If there is no competition for binding, you should be able to quantify the receptors regardless of the ligand presence or absence. Link to comment Share on other sites More sharing options...
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