How To Choose The Correct Antibody For Accurate Flow Cytometry Results

Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. It is by using antibodies conjugated to fluorescent markers that we are able to identify our specific cells of interest and quantitate the amount of our target on the cell.

When I started in flow cytometry, I was immediately taken by the technology, and only later began to appreciate the importance of understanding what my reagents were and how they worked.

With the development and rise of monoclonal antibodies, each lab or group gave them a different name. This name could be the specific clone, where the antibody was harvested, or perhaps the target to which the antibody bound.

You might have attended a talk where one investigator discussed their studies on VLA-4, while a second might have discussed information obtained using Clone 9C10. These are both the same thing, but it was like the wild west out there.

This led to the development of the cluster of differentiation (or CD) nomenclature that we use today. First established in 1982 at the first International Workshop and Conference on Human Leukocyte Differentiation Antigens, researchers from around the world established criteria to classify the different monoclonal antibodies that had been generated by putting clones that targeted the same target in the same CD.

At present, there are well over 370 different clusters of differentiation that have been recognized on human cells. It has also led to the proliferation of those lovely posters that vendors put out, showing the relationship between a CD antigen and the immunophenotype, that is the cells that bind that antigen, and allow us to identify and classify the cells of interest.

For the flow cytometrist it is important to determine how best to select the correct antibody for a given experiment based upon several factors, including:

1. The target
2. The assay
3. Personal experience or preference
4. Lab history
5. Published history/the literature
6. And minor choices such as cost, company, fluorochrome, etc.

Someone entering the field may be very confused as to how to make these choices, especially when they’re asked to design a new polychromatic flow cytometry panel on their own.

Since panel design and antibody choice tend to go hand-in-hand, let’s review some of the important ways to determine how best to choose your antibody, identify the best fluorochrome choice, and build a panel around your hypothesis.

Choose An Appropriate Antibody

Going to the literature is easy, right? Just see what others are publishing and use that. Even better, head over to the website of your favorite vendor. They should have everything you need.

Well, maybe, but maybe not. Let’s take for example, anti-Human CD3. Checking the Human Cell Differentiation Molecules website, they recognize 5 different clones that target human CD3.

To see how prevalent these were in the literature, 2 different metrics were used. The first was to search the Benchsci.com database for these clones and report the published figures for each clone (search term flow cytometry and clone name). The second was to look at the published OMIPs and for each of the human T-cell panels score which OMIP used which clone.

These 2 distinct measurements were quite revealing.

FIGURE 1: CD3 clone use in flow cytometry. Raw data shown in the table below.

Noting that these use 2 different databases and criteria, it is very clear that (1) HIT3a is more represented in one database while UCHT1 is more commonly used in the OMIPs. Which one is better?

The table below shows a bit more detail about some of these molecules. Notice the top 5 are recognized by HCDM, and the bottom 2 were found during the OMIP review.

The solution to this issue is clearly more research.

Don’t just go with what you first find in the catalogue that binds your target. Look deeper and see if there are reasons one reagent is better than another.

Using google-fu,and tools like BenchSci.com and the Antibody Registry, can help you find the targets and where you can get them. Both these resources offer links to publications to make it easier to learn about the reagent in question.

Choose An Appropriate Format

There are several different production methods for antibodies, each with their positives and negatives. These production methods also impact the quality of the binding of the antibody and ultimately the staining of the cells.

1. Polyclonal Antibodies The original way to produce antibodies, injecting an animal with the antigen and collecting the serum is a cheap and easy way to generate multiple antibodies to a given target.

There are disadvantages of this method, especially in the case of using polyclonal reagents in flow cytometry. As shown below in a Western blot assay, it is possible to identify non-specific binding of the polyclonal antibody.

However, in a flow cytometry experiment, it is impossible to distinguish the non-specific staining from true staining, and Isotype controls do not help with this.

Figure 2: Western blot analysis showing the identification of non-specific binding (in red) compared to positive staining (blue). In flow cytometry, it is not possible to easily identify non-specific binding of this nature.

2. Monoclonal Antibodies These are created by fusing an antibody-secreting cell to an immortalized cell to produce a hybridoma cell line that produces a single antibody.

Only those cell lines that secrete high levels of antibody that have high binding efficiency to the target are kept. Köhler and Milstein first created these fusions, leading to the Nobel Prize in Physiology or Medicine in 1984.

Hybridomas have the property where they all secrete an antibody identical to that of the parent cell.

Specifically, these reagents bind to the same epitope, making them ideal for flow cytometry experiments.

One worry about using hybridomas is the concern of cell-line drift, where high-passage number cultures may no longer produce the same antibody as when first characterized.

3. Recombinant Antibodies A newcomer to the flow cytometry world, these are made using recombinant DNA technology, where the genes for a specific antibody are isolated and cloned into a vector for expression.

Since they are genetically engineered, it is possible to change the Fc portion of the antibody, reducing or even eliminating binding to the Fc Receptors on target cells.

These reagents also have the advantage of being less expensive to produce compared to traditional hybridoma techniques, and with the various tools available to manipulate DNA, it is possible to make modifications to the antibody DNA sequence to improve affinity to the target of interest. It is also easier to target those antigens that have been difficult to make by traditional methods.

Bradbury et al., in a commentary in Nature, suggest that moving to recombinant antibodies will improve the quality of these critical reagents.

While monoclonals still dominate the flow cytometry field, as more recombinant antibodies become available, it would seem they will eventually take over the field. This will really help researchers and be a key to improving reproducibility.

How To Start Using A New Antibody

When it comes to using antibodies, one of the biggest issues is the potential for non-specific binding (NSB).

This is where the antibody binds to any cell and increases the background signal. It can also be due to specific, but unwanted, binding of the Fc portion of the antibody to the Fc receptor on the surface of some cell types.

The only way to minimize NSB in an experiment is through proper experimental design and qualification of the reagents.

Fortunately, there are several different steps that can be addressed and 2 — titration and blocking — that specifically relate to antibody use.

1. Titration In the case where there is an excessive amount of antibody in the staining solution, antibodies will bind with low affinity to off-targets on the cell. This leads to an increase in background and a reduction in the loss of signal.

The solution for this is to properly titrate your reagents.

Typically, the titration should start with twice the recommended concentration of reagent, through 6-8 serial dilutions of the antibody. The Staining Index can be used to compare the different dilutions. A typical titration curve is shown below:

Figure 3: Typical titration curve. The optimal concentration on this curve would be defined by the midpoint between the shoulders of the curve, where the staining index begins to decrease.

2. Blocking Using a special reagent to precoat the cells reduces the possibility of the fluorescent antibody binding the cells non-specifically.

Everyone has a favorite reagent (e.g. Fc Block, normal serum, purified IgG) and a favorite concentration, which makes it hard to determine the best approach.

One should perform an experiment where different concentrations of blocking reagent are used and the signal of the negative population is examined to determine which concentration is best.

Andersen and co-workers (2016) did just this, trying to determine both concentration and reagent to use to work with their cells (human PBMCs and monocyte derived macrophages).

They blocked the cells with different reagents at different concentrations, and stained the cells with a labeled isotype control. The median fluorescence intensities (MFI) of the blocked and labeled cells was compared to unstained cells to determine the best reagent.

Based on this data, the authors concluded that the use of purified human IgG was best and least expensive as a blocking reagent. They also recommended that the cells should be blocked for 15 minutes on ice.

Other steps in reducing NSB include the use of viability dyes, adding dump channels to the panel, and gating on similarly sized cells.

With the added emphasis on reproducibility, it is critical to look at every step where experiments can be improved. No single step makes an experiment more reproducible. Rather, it is a process of making changes at each stage that leads to reproducibility.

Antibodies comprise a critical component that needs to be reviewed. As Bradbury et al. in a commentary in Nature pointed out, the global spending on antibodies is about $1.6 billion a year, and it is estimated about half of that money is spent on “bad” antibodies. This does not include the additional costs of wasted time and effort by the researcher using these bad antibodies. Using tools to identify the best reagent to use, considering a switch to recombinant antibodies, and properly validating reagents for use in an assay are 3 steps that will improve the reproducibility of your experiments.

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