How to Choose an Isotype Control

What are isotype controls?

Isotype controls are antibodies with no antigen specificity which are used in immunochemical experiments to control for non-specific interactions between antibodies and the sample. They are most commonly used in flow cytometry and immunohistochemistry experiments where they are particularly useful due to the often high background staining levels. Background staining can be caused by many factors including:

• Binding of antibodies to Fc receptors on target cells. For example some antibodies (e.g. mouse IgG2a) bind to Fc receptors on human leukocytes independently of the primary antibody – antigen interaction.
• Cellular autofluorescence. Some cells and cellular structures contain either proteins or chemicals which naturally fluoresce and this can lead to background staining.
• Non-specific antibody interactions. The primary antibody may bind to non-intended targets meaning that the isotype control can be key in determining if this has occurred.

 

Choosing an isotype control

When choosing an isotype control it is important to keep in mind:

• The host species should be the same as that of the primary antibody
• The isotype (e.g. IgG2a, IgG1) should be the same as the primary antibody
• The isotype control should have the same modifications as the primary antibody (e.g. if the primary is biotinylated so should the isotype control).

 

Using an isotype control

When isotype controls are included in an experiment they should always be used at the same concentration as the primary and following an identical protocol (see our protocols for IHC and ICC). Ideally the isotype control should generate minimal staining, indicating that there is minimal background staining. However this does not prove that the primary antibody is binding specifically!