What are Isotype Controls?
An isotype control is an antibody that does not bind to the antigen, but has similar properties to the primary antibody used in the application. Isotype controls can be utilized as the negative control to help differentiate non-specific background signal from the specific antibody signal. Including isotype controls in some experiments, such as Flow cytometry (FC), Immunohistochemistry (IHC), is important and necessary to improve the experimental operation and data analysis.
Why use isotype controls?
Generally, antibodies are very similar to each other structurally. The majority of an antibody is the “constant region”, shown in Fig.1 This region can help to regulate the antibody mechanism of action (MOA) by binding to Fc receptors. When testing antibodies preclinically, researchers need to control for any variable that might influence the interpretation of experimental results. In fact, the variable region of the antibody determines antigen-specific binding and intended therapeutic effect. Hence, these factors need to be accounted for within the study design. Using a suitable isotype control antibody, which with the same host species, isotype, conjugation, light chain, and concentration as the primary antibody, can account for these added variables and make more precise data analysis.
Fig.1 Structure of antibody.
What applications use an isotype control?
Isotype controls are commonly used in:
- Flow cytometry (FC)
- Immunohistochemistry (IHC)
- Enzyme-linked immunosorbent assay (ELISA)
- Western Blotting (WB)
- Immunocytochemistry (ICC)
Choosing your isotype control
To choose the proper isotype control for your experiment, match the following characteristics of the primary antibody:
- Host species
- Isotype
- Conjugation format:
- Alexa Fluor dyes
- HRP
- Biotin
- Phycoerythrin (PE)
- FITC
- Other formats
Limitations and Recommendations
It is worth noticing that the fluorophore molecules conjugated to a given antibody should be similar in primary and isotype control antibodies. However, these fluorophore could be varied because of different manufacturers. Therefore, it is recommended to purchase both primary antibody and isotype control from the same supplier as far as possible.
With over a decade of experience in antibody productions, engineering, synthetic chemistry, and bio-conjugation, Creative Biolabs offers a wide type of isotype control antibodies now. Choosing the correct isotype control antibody is an important step of most antibody-based experiments. Except for produced antibodies, we also provide viroantibody discovery, engineering and customized services to support customers to achieve their research goals. If you are interested in these services and antibodies, please contact us for more information and a detailed quote.
