What Are 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.
Selecting the Right Isotype Control Antibody
The proper isotype control antibody is needed to correctly interpret experimental results. The following characteristics of the isotype and primary antibodies should match as closely as possible.
- Antibody Species
The primary antibody and isotype control antibody should be from the same host species.
- Antibody Class and Subclass
The primary antibody and isotype control antibody should be of the same class and subclass so that they have the same heavy chain.
Isotype and primary antibodies should have the same conjugation. If the primary antibody is not conjugated, an unconjugated isotype control should be used. Likewise, if the primary antibody is conjugated, the isotype antibody should be bound to the same label.
Applications for Isotype Control Antibodies
Detection of CXCR4 in the Jurkat human acute T cell leukemia cell line by flow cytometry.
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.
Novus Biologicals™ Isotype Control Antibodies
Novus Biologicals, a Bio-Techne brand, provides a large portfolio of isotype control antibodies. Visit the Novus website to view their selection of isotype controls.