Strengths And Weaknesses Of Isotype Controls In Flow Cytometry

Controls are critical for minimizing the effects of the variables in a scientific experiment so that the effect of the independent variable can be accurately measured.

In flow cytometry, there are a host of important controls necessary to properly interpret data generated in these experiments. Some of these controls include compensation, fluorescence minus one (FMO), stimulated and unstimulated, reference, and controls.

When it becomes time to publish, the proper use of these controls is critical in convincing the reviewer and reader that the data has been properly analyzed.

The isotype control is an experimental control where a sample is stained with an irrelevant antibody with the same isotype as the target antibody. Cells are gated and positivity is set based on the background staining of this isotype control.

The use of the isotype control to set negativity remains a topic of discussion and can confuse the novice to flow cytometry, especially when a reviewer may request why these controls were not included in a submitted paper.
Overall, the isotype control is one that is often overinterpreted and can provide little additional information in an analysis.

As Keeney and co-workers stated in their 1998 paper, “Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry — time to let go!”

(Author emphasis).

Clearly, the use of the isotype control is ingrained in the scientific community, and vendors are more than happy to sell you one (or more).

Delving deeper into this specific control and evaluating the strengths and weaknesses can help you to come to your own conclusion on if and when to use an isotype control in your flow cytometry experiments.

1. What is an isotype?

As a B cell matures, it undergoes V(D)J recombination, which results in the production of the B cell receptor (BCR). After selection in the bone marrow, the B cells circulate to secondary lymphoid organs, where they are constantly exposed to new antigens.

When the BCR binds an antigen, the B cell becomes activated and can secrete antibodies as well as generate a memory cell, to provide long-term protection.

Class switch recombination occurs in mature cells upon stimulation, and in the presence of signaling molecules. The variable region of the antibody is retained, while the constant region of the heavy chain is changed, based on the specific signal molecules present at the time. This process is illustrated below.

From: Wikimedia.org

Thus, the isotype is changed.

2. What is an isotype control?

An isotype control is an antibody to an irrelevant target that shares the same heavy and light chain as the target antibody.

For example, the anti-human CD3 antibody HIT3a has the isotype of Mouse IgG2a, κ. Thus, one would look for an isotype control with the same characteristics.

There are several assumptions that are made when an isotype control is used:

1. There is no target for the isotype control antibody expressed on the cells of interest.   The problem with this assumption is that the target of the isotype control is not always known. For example, one can purchase Mouse IgG2a, κ, clone MOPC-173 as an isotype control for CD3 (clone HIT3a). However, the antigen details of the MOPC-173 clone, as described on the BioLegend website (and many other vendor’s sites) states: Thus, the target is not known, it was only selected as an isotype control because of screening against various samples. This does not mean that the target is not expressed on your specific cells of interest, but only that it doesn’t bind in common tissues.
2. The non-specific binding of the isotype control has similar characteristics to the target antibody. There are three factors to consider in antibody binding:
   • Specific binding — this is the binding of the antibody to the target of interest. This is what we are interested in.
   • Fc receptor mediated binding — this is a specific binding of the constant region of an antibody to the Fc receptor expressed on certain cell types. Generally, this is not something we are interested in, and is usually dealt by various blocking methods.
   • Non-specific binding — this is off-target binding of the antigen to any protein in the cell. If the antibody can’t find the target, there is a chance it will bind to another protein. This is often driven by antibody concentration, and one of the critical reasons for titration.Since we don’t necessarily know the target of the isotype control, it is impossible to know what the off-target binding will be. We are left with the fact that the isotype antibody has been ‘tested’ against a standard series of cells and cannot be sure that the NSB of the two reagents will be similar.
3. The fluorochrome to protein (F/P) ratio is the same between the isotype control and the target antibody.   The F/P ratio represents the amount of fluorochrome that is bound to the antibody. In some cases (for large fluorochromes like PE and APC), this is typically in the 1/1 range. However, for smaller fluorochromes, this is not the case, and the labeling of each antibody must be optimized. Thus, unless you know the F/P ratio of both the target antibody and the isotype control, differences in staining and fluorescence could be due to an F/P difference.

3. Should I use isotype controls?

That is the real question.

In addition to the Keeney paper mentioned above, there are several papers that suggest to not rely on isotype controls, including Maecker and Trotter (2006) and Hulspas et al., (2009).

Most recently, an excellent article by Andersen and coworkers (2016) explored the question of how best to block nonspecific binding. Their work was performed on monocytes and macrophages, which contain large amounts of Fc-Receptors.

Figure 1 from Andersen et al., (2017)

This is Figure 1 from Andersen’s paper showing how, in the absence of blocking, the isotype control (IgG1) binds at a much higher level than the specific binding for Tie2, a protein known to be expressed at low levels on monocytes.

However, the plot gets more interesting as the authors explore how best to block this Fc mediated binding of Isotype controls, which they further demonstrate was specific to the Monocyte subset, which is shown in the third figure of this paper. Interestingly enough, the authors demonstrated that this nonspecific binding was only seen with the IgG1 and IgG2a isotype controls.

Figure 3 from Anderson et al. (2016).

The final piece from this paper is the report that different lots of the same isotype from the same vendor showed different binding responses. Again, calling into significant question the use of isotype controls in the setting of gates to determine positivity of a given antigen.

The conclusion from this paper nicely sums up the best practices researchers should be using — and it doesn’t include isotype controls. To quote from Andersen’s paper:

There is one, albeit small, use for isotype controls, also illustrated in the Andersen paper. An isotype control can be used to show that there was poor or incomplete blocking of specific subsets being labeled. That is it.

Looking at all the data and discussions, from the 1998 Keeney paper to the most recent work by Andersen, it is clear that the hypothesis that an isotype control can show the background or nonspecific binding on cells must be rejected. If a reviewer rejects your paper because of the lack of isotype controls, you now have the needed information to rebut those arguments. Likewise, if someone is using an isotype control to set background staining levels, this information can help them realize that they are wasting time and money by using this control for that purpose.

To learn more about the strengths and weaknesses of isotype controls in flow cytometry, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.