Taking a closer look at isotype controls in antibody applications

With the wide variety of experimental techniques relying on primary antibodies, it is important to use both positive- and negative-controls in your antibody applications. We are generally more familiar with positive controls, which confirm antibody reactivity with a known-positive sample. However, we are often less familiar with adequate negative controls. An example of a negative control is an isotype control, which helps to confirm the specificity of a primary antibody.

Isotype controls may serve as a negative control for flow cytometry, immunohistochemistry and western blotting experiments. This control provides a measure of non-specific binding and may strengthen your experimental findings.

The antibody isotype or class denotes differences in the immunoglobulin’s heavy chain. When choosing an isotype control, select one that matches the clonality and isotype of the primary antibody that you are testing.1 For example, for our HIF-1 alpha antibody, researchers purchase our Mouse IgG2B Isotype Control to match the datasheet’s indication that it has an IgG2B isotype and is a monoclonal antibody raised in mice. Additionally, when selecting an isotype control for flow cytometry, it is important that the fluorochrome conjugation is matched.1

 Flow Cytometry: Mouse IgG2B Isotype Control (MPC-11) [NBP2-27231] - Cell surface analysis of 10^6 human PBMC using 1 ug of mouse isotype control. The shaded histogram represents human PBMC alone; green represents mouse isotype control. FITC-conjugated goat anti-mouse secondary antibody was used for this experiment.

While the use of isotype controls may be very beneficial, some caveats should be considered. First, isotype controls may not be used in quantitative analysis (cell counts).1 Second, slight genetic variances between the isotype control and testing antibody may impede a direct match.1 Finally, for flow cytometry applications, isotype controls will not account for fluorescence spillover.2  

Therefore, when using isotype controls, it is important to troubleshoot and carefully select an isotype control antibody that fits your experimental design. Novus Biologicals carries a wide selection of isotype controls for your antibody application needs.

Learn more about Isotype Controls

References

1. Hulspas, R., O'Gorman, M. R.G., Wood, B. L., Gratama, J. W. and Sutherland, D. R. (2009), Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry, 76B: 355–364. doi:10.1002/cyto.b. 20485; PMID: 19575390.
2. Maecker, H. T. and Trotter, J. (2006), Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry, 69A: 1037–1042. doi:10.1002/cyto.a.20333; PMID: 16888771.