Isotype controls are antibodies used as negative controls to determine background signals caused by nonspecific, non-epitope-driven binding. Causes of unwanted background may arise from artefacts, debris, nonspecific binding to cellular components, and certain cell types, such as some leukocytes that express immunoglobulin-binding Fc receptors. In choosing an isotype control for your immunostaining or flow cytometry experiment, one should consider the control’s similarity to the primary antibody and some limitations.
The Ideal Isotype Control:
The isotype control is similar to the primary antibody, but are raised against an antigen that is not present in the experimental sample. The control should match the host species, antibody class, and subclass, and conjugate of the primary antibody. For example, a rat IgG2a primary antibody conjugated to APC would call for a rat IgG2a APC isotype control. A matching light chain class (kappa or lambda) would also be more ideal.
Limitations:
The number of fluorophore molecules conjugated to a given antibody (known as the F/P ratio) should also be similar in the primary and isotype control antibodies. However, these ratios can vary between different manufacturers. Thus similar antibody conjugates from different sources may have varying levels of staining that should addressed if it arises. Isotype controls cannot account for spillover of emission spectra from multiple fluorescent sources. A multi-color assay will ideally require multiple isotype controls. Isotype controls cannot be used to determine negative populations.
Recommendations:
Because the way conjugates are attached to antibodies may vary per manufacturer, it is recommended to purchase both primary antibody and isotype control from the same supplier. Some companies may even recommend an accompanying isotype control product along with a given primary antibody. In addition to isotype controls, other controls should also be considered, such as compensation controls and fluorescence minus one (FMO) for multicolor assays, as well as unstained cells to account for autofluorescence.
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Relevant Literature:
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Hulspas, R., O’Gorman, M. R. G., Wood, B. L., Gratama, J. W. & Sutherland, D. R. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom 76, 355–364 (2009).
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Maecker, H. T. & Trotter, J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037–1042 (2006).
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McCracken, J. When To Use (And Not Use) Flow Cytometry Isotype Controls. ExCyte
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