Isotype Controls in Flow Cytometry

When you look at the structure of your fluorophore-conjugated primary antibody, it is composed of the variable region that can recognize the particular antigen and the fragment crystallizable region (Fc region) that belongs to the host species where the primary antibody has been raised. The isotype of immunoglobulin (antibody) is determined by the differences of amino acid sequences in the Fc region of antibody heavy chains. Therefore, the isotype of your primary antibody is determined by the host species. 

What happens in a Flow Cytometry staining when you incubate your cells with the primary antibody? For example, you have a fluorophore-conjugated primary antibody that is raised in goat (the host) against X antigen of a mouse, the Goat anti-mouse X antibody of IgG2a isotype. When incubating this antibody with the mouse cells, two major reactions will occur inside the tube, which would result in fluorescence staining. They are as follows:

1. The primary antibody will recognize the X antigen specifically and will result in positive staining or fluorescence.
2. The Fc region of your primary antibody that belongs to the host species where it has been raised, in this case, the goat, will bind non-specifically to the Fc receptors present in the mouse cells (B cells, macrophages, etc.), resulting in non-specific or background fluorescence. In addition, the goat-originated constant region may produce other non-specific protein interactions, which might also result in background fluorescence. 

To uncover this non-specific or background staining, the straightforward way is to use a fluorophore-conjugated non-specific antibody, which is consisted of the Fc region of the host species, with the same isotype, in this case, the goat immunoglobulin, IgG2a (Goat IgG2a). This is called isotype control antibody. The level of fluorescence (signal intensity) resulting from staining with the isotype control will be the non-specific binding or the background fluorescence. To get rid of Fc mediated background staining, you can use an appropriate Fc receptor blocking antibody before the primary antibody. 

Basically, isotype controls should closely match the properties of the primary antibody and be used under identical experimental conditions to best determine the presence and extent of non-specific binding. Isotype controls should be from the same host species, class, and subclass (isotype) and used at the same working concentration as the primary antibody. The isotype control should also have the same conjugate when using a labeled primary antibody. Moreover, as the fluorophore conjugation to the antibody (known as the F/P ratio) can vary between suppliers, it is best to purchase the isotype from the same supplier as the primary.

Taken together, isotype controls can be concluded to serve two functions. Firstly, it would confirm the specificity of your primary antibody. Secondly, it will show you the fluorescence resulting from Fc receptor-mediated binding and other non-specific cellular protein interactions.