Isotype Control Antibodies

Isotype control antibodies are commonly used negative controls. Immunoassays utilize immunoglobulins to detect specific analytes. These immunoglobulins, however, can also bind to cellular proteins that are not their target antigen, generating non-specific signals. Additionally, cells and tissues used in immunoassays can exhibit natural fluorescence. To differentiate between specific signals and background staining, either due to non-specific binding or autofluorescence, isotype antibodies are used as experimental controls. Isotype controls are antibodies that match the species, isotype, and conjugation of the primary antibody, but are specific to analytes that are not found in sample type being analyzed, such as Keyhole Limpet Hemocyanin (KLH) and Hen Egg Lysozyme (HEL). They provide a means to measure background staining and indicate whether further experimental optimization is needed.

Isotype control antibodies are mostly used in flow cytometry experiments. In flow cytometry, antibodies can bind non-specifically to surface Fc receptors, producing background staining, which can confound the results. Background staining is especially problematic when analyzing rare cell populations or lowly expressed proteins, or when building multicolor flow cytometry panels. Using an isotype antibody that matches the species, class, conjugation, and concentration of the primary antibody allows for the differentiation between the specific signal and non-specific background staining. Isotype control antibodies can also be used as blocking and protein coating agents for other applications including ELISA, Western blot, immunohistochemistry, and immunocytochemistry.