Isotype controls are primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application. Isotype controls are used as negative controls to help differentiate non-specific background signal from specific antibody signal. Depending upon the isotype of the primary antibody used for detection and the target cell types involved, background signal may be a significant issue in various experiments.
Causes of background staining associated with primary antibodies include:
- binding to Fc receptors on target cells
- nonspecific antibody interactions with cellular proteins, carbohydrates, and lipids
- cellular autofluorescence
Selecting an isotype control
Isotype controls should be matched to the primary antibody’s host species and class, including light chains (e.g., IgG2 Lambda, IgA1 Kappa). If using directly labeled primary antibodies, it is important to use the same conjugate to avoid differences in detection.
Applications for isotype controls
Although isotype controls are mainly used in flow cytometry, they can be used as standard blocking agents and protein coating agents for other applications including immunofluorescence, immunocytochemistry, western blotting, and ELISA.