Operation Titration: Tips for Titrating Your Antibodies

To get the best results out of your flow cytometry experiments, titration of your antibodies is a crucial yet often overlooked step of the workflow. Simply put, titration is the testing of your antibody at a range of concentrations to determine which gives you the best signal-to-noise ratio. 

Titrating your antibodies can help you avoid wasting precious resources when less product could be used to achieve the same results. Not only can titration save you money, but it could also improve the quality of your data. Picture a Goldilocks and the Three Bears type of situation (Figure 1). Using a high concentration of your antibody is too much — excess antibody may bind nonspecifically to proteins other than the target and create background signal. However, a low concentration of your antibody is too little — this may result in an inability to detect your target protein. But, by the power of titration, you can find the antibody that is just right for your experiment!

In this blog, we discuss best practices for titrating your antibodies for flow cytometry.

Fig. 1. A depiction of the “Three Bears” of antibody concentrations.

Don’t Wait to Titrate

Ideally, you should titrate each new antibody you purchase before performing any experiments with it. More often than not, antibody suppliers will suggest a dilution or concentration range for your antibody. While this can be a good starting point, you should still titrate the antibody yourself, as your experiment setup will likely differ from that of the supplier.

For example, if a CD127 antibody has been titrated via the staining of memory T cells, which express high levels of this marker, and your target population is naïve regulatory T cells (Tregs), which express low levels of the protein, your requirements could differ significantly, and the recommended concentration may not be enough for your purposes (Liu et al. 2006).

You should titrate not only when using an antibody for the first time but also when you receive a vial of a new batch. Although each batch should work similarly, small differences can affect the product's performance, so it’s good practice to check that a new batch works like the old one and adjust accordingly.

Lastly, while the focus of this blog is on flow cytometry, antibodies can also be used for a range of other applications, such as western blotting, ELISA analysis, and immunohistochemistry. You should perform a titration in each new application, even if you are using a familiar antibody. Remember, the optimal concentration for one application may not be the same for another!

Titration Best Practices

So, we know why and when to titrate, but now for the big question: how exactly do we titrate?

First and foremost, it’s important to keep other experimental factors, such as the cell type, the secondary antibody used (if required), and the staining volume, consistent between the titration and upcoming experiments that use the titrated antibody. If, in the future, you want to use the same antibody on a different cell type or under different conditions, it’s best practice to titrate again. If you plan on performing a multiplex experiment, make sure you titrate all your antibodies separately.

To perform your titration, you need to stain your cells of interest with a range of concentrations of your test antibody. Ideally, you should test various dilutions above and below that recommended by the supplier. For example, if the recommended dilution is 1:200, a good dilution series to test would be 1:50, 1:100, 1:200, 1:350, and 1:500. You should also include an unstained control to determine the level of background or autofluorescence in your samples.

When preparing your samples, it’s important to consider the specifics of your experiment. For example, if your target cell is a rare cell type, you may need to work with higher cell numbers per sample to acquire a sufficient amount of your cell population. But remember to use the same number of cells per dilution!

You may also need to include other markers in your titration experiment to accurately identify your population of interest. Additionally, it’s good practice to include a viability dye to remove dead cells. Dead cells typically have greater autofluorescence and increased non-specific antibody binding, which can lead to false positives and skew your results.

Once you have acquired your data, you can determine the optimal dilution by calculating the stain index of each sample. The stain index is the ratio of separation between a positive and negative population. This can be calculated using the equation below (Figure 2).

Fig. 2. Calculation of the stain index. The stain index is the mean fluorescence intensity of the positive population (green) minus the mean fluorescence intensity of the negative population (black) divided by two times the standard deviation of the negative population. SD, standard deviation.

Figure 3 shows a real life example of the stain index in action, used to determine the optimal concentration for Bio-Rad’s CD4 SBUV795 antibody. Perform this calculation on all the concentrations in your titration experiment to establish the best signal-to-noise ratio.

Fig. 3. Stain index calculation for Mouse Anti-Human CD4 Antibody conjugated to SBUV795. SBUV, StarBright UltraViolet.

The optimal dilution is the one that gives the highest stain index. In Figure 4, you can see an example of a stain index plot where the stain index is plotted against concentration. For this antibody the optimal concentration is 0.5. 

Fig. 4. Stain index plotted against concentration.

Lastly, our final piece of wisdom is to always record the product code and batch number of the antibody you’ve used in an experiment. This will allow you to compare its performance over time and assess any differences between batches. Additionally, you will need this information if you need to request product information from the manufacturer for troubleshooting.

Following best practices for antibody titration will allow you to maximize the detection potential of your experiments, get more reproducible results, and reduce the costs of your experiments. In summary: there’s no debate, you must titrate!