Isotype controls are negative controls that help differentiate the level of non-specific background signal from the specific signal produced by the primary antibody. Typically, this background signal is the result of immunoglobulins binding non-specifically to Fc receptors on target cells; it can also be due to non-specific antibody interactions with cellular proteins, carbohydrates, or lipids as well as by cellular autofluorescence.
Isotype controls are most commonly used in flow cytometry, but may also be used as standard blocking agents and protein coating agents for other applications including immunofluorescence, immunocytochemistry, western blotting, and ELISA. They are typically normal serum- or myeloma-derived antibodies of an unknown specificity or raised against an antigen that is presumed to be absent in the sample being evaluated.
Selecting an Isotype Control
Isotype controls should be matched to the primary antibody’s host species and class, including light chains (e.g., IgG1 Lambda, IgM Kappa). It is also necessary to make sure that the isotype control is conjugated to the same fluorochrome or label as the test antibody. It is not sufficient to use one with a spectrally similar fluorochrome.
For example, if your primary antibody is a Mouse (IgG2b) Anti-Human XYZ-APC, you will need a Mouse IgG2b-APC isotype control. It is typically recommended that the isotype control be used at the same concentration as the primary antibody.