Obtain reproducible results through antibody titration

Despite their widespread scientific, clinical and commercial use, there is frustration around difficulties caused by poorly validated antibodies and batch-to-batch variability.^1,2

This can lead to abandoned projects and wasted time, money and samples, and is considered the major driver of the ‘reproducibility crisis,’ a growing realisation that results of many biomedical experiments cannot be reproduced.²

The use of the improper dilution of the antibodies, which can generate unwanted background, is one of the major sources of poor-quality results in flow cytometry. The recommended dilution of the antibody given by the vendor is not always appropriate for the specific assay conditions.³

Antibody concentrations that are too low can make a truly positive population appear (falsely) negative and antibody concentrations that are too high can make a truly negative population appear (falsely) positive.⁴

To overcome this, an important aspect of designing multicolour flow cytometry panels is antibody titration.⁵

What is antibody titration?

Antibody titration is not only necessary to determine whether reagents work and perform optimally, but also to define the optimal antibody concentration that ensures the best separation of positive population from the background.

The optimum antibody concentration is selected by determining the maximum stain index for an antibody.

It is influenced by several factors, such as staining temperature and duration, as well as the type of fixation/permeabilisation buffer; therefore, it is recommended to perform the antibody titration under the same conditions as the final experiment.

Titrations should be conducted for each antibody in the panel and repeated to ensure the accuracy and reproducibility of the titration experiment.

Protocol to establish optimal antibody concentration

As well as your centrifuge, flow cytometer and round-bottom tubes, you will need:

• — Antibodies of interest labelled with fluorochrome (at a concentration 4 x the manufacturer’s recommendation; can be higher or lower if desired)
• — Phosphate-buffered saline without calcium or magnesium (containing 1% bovine serum albumin)
• — Sample: cell suspension (1-5 × 106 cells/ml); the cells should be prepared, unfixed or fixed, in the same way as they will for the multicolour experiment

Prepare antibody serial dilutions⁷

• 1. Label each of your tubes from 1 to 9
• 2. Add 50 µl of staining buffer to each tube
• 3. Add 50 µl of antibody at 4 x the manufacturer’s recommendation to the first tube
• 4. Mix well and transfer 50 µl from tube 1 to tube 2
• 5. Repeat this step until tube 7 contains 50 µl, then discard this from tube 7

Add cells⁷

The cell preparation must contain a mix of cells that are positive for the epitope and cells that are negative for the epitope.

• 1. Add 100 µl of the 1-5 × 106 cell/ml suspension to all tubes and mix well
• 2. Incubate for 30 minutes at room temperature in the dark (or follow recommended conditions for your antibody)
• 3. Wash the cells by adding 2 ml of staining buffer, centrifuging for 5 minutes at 300 x g, 4°C, removing the supernatant, vortexing the pellet and resuspending in 200 µl staining buffer.

Following analysis of the data acquired through flow cytometry, a titration curve can be constructed comparing the stain index to antibody concentrations; the maximum stain index indicates optimal antibody concentration.  

Warning: if antibody affinity is low, the titration curve will have no clear saturation plateau, meaning optimal antibody concentration is difficult to obtain; therefore, they are extremely prone to false-negative or false-positive result.

Discover more flow cytometry protocols.