The Art of Selecting Isotype Controls

by Creative Diagnostics Manufacturer & Supplier

What is an isotype control

An isotype control is a type of negative control which is designed to measure the level of non-specific background signal caused by primary antibodies. Considering the tissue type of the sample used, the results might vary significantly.

Flow cytometry experiments will frequently need isotype controls, and so does immunohistochemistry, but less frequently.

The importance of selecting an appropriate isotype control

For almost all flow cytometry experiments, the choice of a proper isotype control could be decisive. Due to the features of no specificity for target cells, an isotype control antibody is commonly used in cytometry experiments. At the same time, isotype control antibody retains all of its non-specific characteristics of the antibodies used in an experiment.  Based on this, the reasons for us to use isotype control are concluded as follows:

On one hand, to confirm the specificity of primary antibody binding; on the other hand, to rule out non-specific Fc receptor binding or other cellular protein interactions.

Isotype controls in flow cytometry experiments: surface staining and intracellular staining

1.     Surface Staining

This is a very common application as they are optimally developed for cell surface staining protocols and help assess the level of background staining inherent in cell-antibody binding assays. Here, we’d like to mention two tips from Creative Diagnostics experts for the successful use of isotype controls:

·           The isotype control antibody is better to ideally match each primary antibody’s host species and isotype since there are variations in non-specific binding.

·           It is better if the isotype control and the primary antibody have the same antibody concentration. We can’t expect isotype controls to be available for every antibody.

2.     Intracellular Staining

It is a great tool to optimize flow cytometer settings and establish specific data sets for autofluorescence. Many experiments have found that the use of isotype controls here usually gain inconsistent staining, including inherent differences in the amino acid composition of the two antibodies, or different F/P, which is inconsistent amount of fluorophore conjugated to the isotype control compared to the experimental antibody.  Activation of cells may also alter the staining patterns of isotype control antibodies.  Therefore isotype controls are preferred to be used by combining with other negative controls or using unstimulated cells, inherently negative population cell.

At Creative Diagnostics, scientists made great efforts to minimize inconsistencies by using proprietary technologies and consequently generating high-quality isotype control antibodies within a standard range.

Contact

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Creative Diagnostics

Tel: 1-631-624-4882 
Fax: 1-631-614-7828 
Email: info@creative-diagnostics.com

Web: http://www.creative-diagnostics.com/CommonTypeList_435.htm

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Created on Dec 31st 1969 18:00. Viewed 0 times.

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