Isotype Control Antibodies: Overview & Usage

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Using appropriate controls for accurate interpretation of results is critical for producing scientific results. Negative controls are one way to provide additional confirmation of staining that is observed in immunoassays such as Flow Cytometry and Immunohistochemistry. Using negative controls can help distinguish between non-specific background staining and specific antibody detection. An isotype control is a type of negative control that can help researchers distinguish if the results observed are specific and reduce the risk of false-positive results.

R&D Systems offers a wide variety of isotype control options that cover multiple species, isoforms, and conjugates to match primary antibodies.

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What Is An Isotype Control?

An isotype control is an antibody used as a negative control to determine the amount non-specific background staining that can be observed. Isotype control antibodies have the same immunoglobulin isotype as the primary antibody in the sample being analyzed, but is specific to a known irrelevant target, such as keyhole limpet hemocyanin (KLH). Non-specific Fc binding can occur, and it is important to determine how much of the observed results are specific compared to non-specific binding of the antibody. Cells can also have a natural fluorescence that cannot be distinguished without a negative control. By measuring background staining, isotype controls will help indicate if further optimization is required or if the sample is adequately blocked and washed.

Isotype Control antibody

How Do I Select An Isotype Control?

An isotype control should match the primary antibody as closely as possible. Select an isotype control that has the same host species, isotype, and conjugate as your primary antibody. For example, if the primary antibody has the host species as mouse, an IgG₁ isotype, and conjugated to phycoerythrin, the isotype control would also have a mouse IgG₁ isotype and conjugated to phycoerythrin. If the primary antibody is unconjugated and a secondary antibody is being used, the isotype control should still match the primary antibody and be unconjugated. Having the isotype control match the primary antibody as much as possible will help determine that amount of non-specific staining that may be caused from the primary antibody.

Isotype Control antibody

How To Use An Isotype Control?

When using an isotype control, the primary antibody and isotype control should be handled in identical conditions. The same protocol is used with the primary antibody as well as with the isotype control. This includes using the same concentration for the isotype control and primary antibody. When using a secondary antibody, the isotype control is added in the same manner as the primary antibody. Samples should be treated the same way with both primary and isotype control antibodies. By eliminating as many experimental differences as possible, an accurate reading of the assay can be accomplished.

Detection of CXCR4 in Jurkat Human Cell Line by Flow Cytometry with Mouse IgG2B Isotype Control Antibody