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In a Flow Cytrometry, one can add an Isotype Control Antibody to allow another antibody to bind more specific to the cells.

My question is, how can the Isotype Control Antibody add specificity to the other antibody? My thought was, that the Isotype Control Antibody binds to all the antibody receptors, but with a "weak" binding. When the other antibody (e.g. CD73) tries to bind, it can make a stronger binding with the CD73 antibody receptor, kicking the Isotype Control Antibody off it.

However i'm entirely not sure this is correct... Could anyone explain how the Isotype Control Antibody helps the other antibody (eg.g CD 73) to bind more specific?

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migrated from Aug 28 '12 at 12:57

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I don't believe that the Isotype Control Antibody makes the antibody bind more specifically but rather ensures that your signal is specific to your antibody.

The Isotype Control Antibodies tend to bind to the non-binding domain of the antibody, typically the constant (Fc) domain. By measuring the concentration of the control antibody, you can determine what percentage of your light signal comes from your primary antibody and not from non-specific binders to your light source.

This is very similar to having secondary antibodies in your assay.

Random link showing that I'm not making stuff up

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This is correct. It adds credibility that your experimental primary antibody is actually binding what you think it binds. – user560 Nov 7 '12 at 1:11

While bobthejoe/leonardo's answers are correct, you also need to be aware of the quality and limitations of isotype controls. You need to pick your source very carefully, as extensive testing (at my former company) has shown that some isotypes stick non-specifically under certain conditions or with certain samples. For example, we found that one common isotype from a major manufacturer (I don't remember who, it might have been Jackson) stuck non-specifically to HeLa cells by immunofluorescence and flow cytometry (the chemistry in the two assays is very similar) when used with common IF and FC protocols. You should also run no-antibody and secondary-only controls to ensure that your samples are not auto-fluorescing and that the secondary antibody is not binding non-specifically to your sample. Finally, ensure that the control exactly matches the isotype of your primary - don't use a rabbit polyclonal isotype to control for a rabbit mono, don't mix IgG subtypes, etc. Good luck!

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