Isotype Control Antibodies

1. Overview

Isotype control antibodies refer to the immunoglobulin sharing the same species, subtype and dose as the primary antibody. It's mainly applied in removing the background signal caused by non-specific binding.

2. The Purpose of Isotype Control

We know that the antibody structure is divided into a constant region and a variable region. The variable region determines its specific binding to antigens. The constant region where antibodies play the regulatory role by the binding between FC site and FC receptor from various cells usually consists of conserved region. The sequences of isotype antibodies are usually the same.

During biological experiment, setting isotype control antibodies group can effectively eliminate the background signal caused by non-specific binding and accurately reflect the specific binding between primary antibody and antigen. The experimental data are more reliable. Moreover, the research article using relevant data is more easily accepted by high IF journals.

3. Applications 

FC

Isotype control is one of the most commonly used negative controls in flow cytometry. It simulates non-specific binding and staining of FcR or other proteins, and also can set the voltage of flow cytometry to efficiently save experimental cost(Figure 1).

Figure 1.  Isotype control signal(Blue) is weaker than target antibody signal(Red), showing the antigen-antibody specific binding.

Figure 2. The staining of control group only includes the heavy chain band (55kd), showing the non-specific binding between the antibody and the sample.

IP

The isotype control sample can only detect the heavy chain band of the antibody(Figure 2).

IF and IHC

The staining signal of isotype control is weaker than specific antibody while reacting with the target protein of the sample(Figure 3).

Figure 3.  Isotype control is unstained (left). Nucleolus is obviously stained (right), showing the specific staining between antibody and sample.

ELISA

When ELISA assay detects the specific antibody, isotype control can exclude non-specific binding.

4. How to choose an isotype control antibody?

• The same species: Mouse, Rat, Hamster
• The same subtype: IgG1, IgG2a, IgG2b, IgG3, IgM
• The same labeling: FITC, PE, PerCP/Cyanine5.5, PE-Cyanine7, APC etc.
• The same concentration

Notes: If you don't have the exactly matched isotype control antibody, you can choose it in accordance with the same species, fluorescence labeling and IgG type, following the priority below: different subchain→different subtype→same species.

5. Recommended Products

REFERENCES

[1]Measurement of isotype-specific antibody responses to Aujeszky's disease virus in sera and mucosal secretions of pigs, PMID: 1315087.
[2]Serum and intestinal isotype antibody responses to Wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies, PMID: 10374959.