Antibodies bind specifically to unique antigens, but they can still have interactions not involving the antigen-binding domain that produce background signal. These off-target interactions include the Fc region of the antibody binding to Fc receptors or other cellular components, as well as interactions of the antibody’s conjugate (such as a fluorophore) with proteins and molecules in the cell or sample. Isotype control antibodies match the host species and class of the test antibody but are generated against irrelevant antigens not present in cells or samples of interest, and are therefore useful for characterizing background signal caused by off-target binding. The diagram below summarizes important features to consider when choosing the right isotype control. Use the following table to browse isotypes by host species and antibody class.
The isotype must have the same conjugate as the test antibody. Additionally, the protein concentration and fluorophore:protein ratio (if the conjugate is a fluorophore) must match.
The isotype and test antibody must be from the same host species and of the same class (i.e. mouse IgG2b), which impacts how it interacts with Fc receptors or other cell components.
The only difference between the isotype and test antibody should be antigen specificty: isotype controls do not specifically bind to any antigens present in cells/samples of interest.