Isotype Controls

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.

Conjugate 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. Antibody species and class 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. Antigen specificity 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.

FcR blockers

If an isotype control produces high background signal, it may be due to Fc receptor (FcR) binding to the Fc portion of antibodies. Samples containing high frequencies of cell types expressing FcRs (granulocytes, B cells, macrophages, dendritic cells, and others) may require the use of FcR blocking reagents. Utilize Human TruStain FcX™, a blend of human IgG antibodies that use their Fc portions to block FcRs, and Mouse TruStain FcX™ PLUS, an antibody specifically directed against CD16 (FcγRIII) and CD32 (FcRγII) through its antigen-binding domain, to prevent background signal from FcR binding.