What is an isotype control?
During the antibody staining procedure, it is important to note that the primary antibody can bind not only to its target antigen but also to non-specific molecules such as reactive epitopes or Fc receptors present on cell surfaces. This non-specific antibody interaction (off-target interaction) can produce a background signal which results in an alteration of the experimental results. Therefore, when you are using a primary antibody for the correct detection of an antigen, it is important to distinguish between the specific signal and the background signal produced by the antibody. For this reason, in the place of the primary antibody, another antibody known as an isotype control should be used. The isotype control is an antibody raised against an antigen that is not expressed in the analyzed sample. The isotype control differs from your primary antibody only for the specificity of the antigen and does not specifically bind to any antigens present in the sample of interest.
Why are isotype controls important?
Isotype controls are very important in order to ensure that the primary antibody staining is specific rather than non-specific background signal. An isotype control, also known as a negative control, is a primary antibody that does not bind to any antigen in the sample but matches all the properties of the primary antibody being used, such as: host species, isotype (and subclass), and conjugation. In research experiments such as flow cytometry (FC) and immunohistochemistry (IHC), there is a high risk of background staining, and an isotype control should be used to ensure the staining patterns are interpreted correctly. It is not possible to predict the unspecific binding of a primary antibody to non-targets molecules, as such, an isotype control is necessary in order to evaluate the contribution of non-specific background signal to the staining results and to distinguish between non-specific and specific antibody staining.
What causes non-specific background staining?
Generally, you can have a background signal when your primary antibody binds non-specifically to the Fc receptors expressed on cells, such as: B cells, macrophages, monocytes, and dendritic cells. Additionally, the primary antibody can also bind non-specifically to lipids and carbohydrates and can interact with off-target proteins or debris present in the sample.
How to choose an isotype control?
An isotype control antibody must match the properties of the primary antibody being used. It must be raised in the same host species, be of the same isotype and Ig subclass, and have the same conjugation. For example, if you are using a mouse IgG1 monoclonal antibody (PE conjugated) you should use a mouse IgG1 (PE conjugated) isotype control.
What experimental conditions should you consider?
The isotype control antibody should be used in place of the primary antibody and with the same experimental conditions, including: working concentration (μg/ml), temperature, incubation, and detection method. It is also important to use an isotype control from the same supplier as the primary antibody, as the isotype control should ideally have an equivalent label to antibody ratio with respect to the primary antibody, and conjugation methods and the resulting number of fluorophore molecules conjugated to a particular antibody can vary between suppliers.
How can you reduce non-specific antibody binding?
You can reduce non-specific antibody binding by blocking Fc receptors, for example, by adding an excess of BSA to the buffer. Additionally, you can reduce the amount of antibody being used, by reducing the working concentration or performing a titration of the primary antibody, and you can also gate out dead cells in flow cytometry experiments by using live / dead makers.
