Isotype Controls: Negative Isotype Controls

Overview

Isotype controls are a type of negative control designed to measure the level of non-specific background signal caused by primary antibodies, based upon the tissue type of the sample. Usually, the background signal is the result of immunoglobulins binding non-specifically to Fc receptors present on the cell surface. For example, antibodies raised in mice, particularly those of the IgG2a isotype, will bind strongly to some human leukocytes regardless of the test antibody specificity. In this case you would need a mouse IgG2a isotype control for use with human cells or tissues.

Isotype controls are most commonly used in flow cytometry experiments, but may also be used in immunohistochemistry.

Selecting an Isotype Control

Isotype Controls for Flow Cytometry- Determine Specific Binding | Download PDF

Typically, an isotype control is matched to the host species and isotype of your specific primary antibody. Sometimes, it is an antibody that has been raised against an antigen that is not normally expressed in the target tissue, e.g. DNP, but this is not always the case.

When using directly labeled primary antibodies, it is also necessary to make sure that the isotype control is conjugated to the same fluorochrome or label as the test antibody. It is not sufficient to use one with a spectrally similar fluorochrome. Bio-Rad offers isotype control cocktails for commonly used dual and tri-color staining combinations.

Recommendations

Recommended isotype controls are usually found at the bottom of Bio-Rad product datasheets. In addition, we have added a table below, which you can use to filter our products by various attributes.

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Resources

For assistance with your experiments, visit our resources section for detailed protocols, tips, guides and educational articles. For more information or assistance with choosing an appropriate isotype control, please contact our technical support specialists.