The section of the above paper focusing on isotype controls summarizes the problems with their use very clearly. The section on isotype controls summarizes the problems with the use of isotype controls very clearly…įlow cytometry controls, instrument setup, and the determination of positivity.Īdditionally, the following paper presents options for controls in several categories, the options available, and pros and cons of each option…Ĭonsiderations for the control of background fluorescence in clinical flow cytometry. The following paper presents options for controls in several categories, the options available, and pros and cons of each option. If you spend any time browsing the Purdue Cytometry list, you’ll see these same arguments presented in threads about isotype controls. With some suggesting they be left out of almost all experiments. Moving Beyond Isotype Controlsįor these reasons, many in the field are moving beyond the isotype control. There is even a case to be made that differences in the amino acid sequence of the variable regions of both the light and heavy chains might result in variable levels of undesirable adherence in isotypes versus your antibody of interest. This, unfortunately, makes the manufacture of ideal isotype controls highly impractical. F:P is a measurement of how many fluorescent molecules are present on each antibody. In order to be useful, the isotype control should ideally be the same isotype, both in terms of species, heavy chain (IgA, IgG, IgD, IgE, or IgM) and light chain (kappa or lambda) class, the same fluorochrome (PE, APC, etc.), and have the same F:P ratio. This can be Fc mediated binding, or completely nonspecific “sticky” cell adhesion. What it does do is give you an estimate of non-specific (non-epitope-driven) binding. Not all of these can be directly addressed by this control (such as cross-reactivity to a similar epitope on a different antigen, or even to a different epitope on the same antigen).
The idea is that there are several ways that an antibody might react in undesirable ways with the surface of the cell. Isotype controls were classically meant to show what level of nonspecific binding you might have in your experiment. Most importantly, why do reviewers keep asking for them when they review papers containing flow data? What are they, why and when do I need them? Are they of any use at all, or just a waste of money? They are still very often included by some labs, almost abandoned by others, and a subject of confusion for many beginners. Isotype controls were once THE negative control for flow cytometry experiments. When used to label cells, those that showed binding to the isotype, would be excluded as they represented the non-specific binding of the cells. The concept of this control is that an antibody targeting a protein not on the surface of the target cells, has the same isotype (both heavy and light chain) as the antibody of interest. This led to many research groups using a control called the Isotype control.
CONTROLS FOR ANTIBODY CROSS REACTIVITY HOW TO
The question has always been how to identify and control for the nonspecific antibody binding observed. Further, as cells die, and the membrane integrity is compromised, antibodies can non-specifically bind to intracellular targets. They can also bind to cells in a nonspecific manner, where the FAB portion binds to a low affinity, non-specific target. Antibodies can bind to cells in a specific manner – where the FAB portion of the antibody binds to a high-affinity specific target or the FC portion of the antibody binds to the FcR on the surface of some cells.
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