Isotype controls are antibodies of the same class (isotype) of immunoglobulin as the specific antibody, but
are either myeloma-derived antibodies of unknown specificity, or are raised against an antigen that is presumed not to be present on or in the cells under study. The ideal isotype control should match the specific antibody not only in heavy chain (IgA, IgG, IgD, IgE, or IgM), subclass and light chain (kappa, lambda) class but also in fluorochrome type and number of fluorochrome molecules per immunoglobulin (F/P ratio). Isotype control antibodies are used for the estimation of non-specific binding of target primary antibodies to cell surface antigens. They are particularly useful for assessing the up or down-regulation of a specific surface marker. They can also give valuable information when dealing with samples that are burdened with poor viability and large amounts of cellular debris.
Selection of an appropriate isotype control is important. The problem with using isotype controls to set gates blindly is that many antigens do not show bimodal expression patterns that are either "on" or "off. Hence, blindly using isotype gates or background, can only lead down the path marked "Artifact. An Isotype control, in some cases, does not work and can in fact lead to erroneous estimations of the target subpopulation. In the ideal situation of matched isotype and subclass, matched F/P ratio and concentration, there may still be differences in background binding characteristics of a negative control. These are limitations inherent in the use of an isotype control. If their use is approached with an understanding of these limitations then they can be particularly useful.