Methods and Principles from our Scientific Staff

Background

Titration is the process of identifying the correct concentration of antibody to use for a given assay. This ensures the antibody performs within acceptable parameters including reducing negative effects and waste. Titration for flow cytometry is used to determine the antibody amount and concentration resulting in the highest signal of the positive population along with the lowest signal of the negative population. It is also the best way to eliminate nonspecific binding of an antibody.

Why Do It?

The precise amount of an antibody is essential to proper titration.

Too much antibody will result in an increase in the background non-specific (or low affinity) binding, reducing the sensitivity of the fluorescent measurement.

Too little antibody will result in a decrease in the positive signal, again reducing the sensitivity of the fluorescent measurement.

Figure 1. (Left) As concentration of antibody increases there is greater separation of positive and negative cells based on fluorescence intensity.

Leinco Product C119, Anti-Human CD8 (Clone: UCHT-4) – FITC