Antibody Validation for Flow Cytometry

Antibody validation is to confirm (or refute) that the antibody is selectively detecting the target-of-interest in your assay and sample-of-interest. The approaches available broadly map onto the five pillars of antibody validation (see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity of an antibody for flow cytometry experiments. This will include a discussion of the Human Leucocyte Differentiation Antigens (HCDM) workshop approach, which focuses on validating antibodies against markers of the human blood leukocyte populations, as well as a pragmatic approach to determining the selectivity of antibodies for other sample types and for non-surface (intracellular) antigens.

Unfortunately, antibodies frequently do not work as assumed (Ayoubi et al., 2023). Additionally, their performance depends on the specific protocol used and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is, an antibody shown to perform well in one set of conditions may not perform as well in other conditions. For these reasons, it is essential to test the selectivity of your antibody. But testing is not one size fits all. The approaches used will be different depending on the sample and target type.

To start the validation process, one must first confirm that a candidate antibody can detect the antigen of interest in the given protocol. This requires use of one of the five pillars of antibody validation, and it is recommended to screen multiple candidate antibodies when possible. If cost is prohibitive, consider an approach that prioritizes more reproducible/renewable reagents, such as recombinant or hybridoma-derived monoclonal antibodies. It is also important to seek out supportive data on candidate antibodies from the manufacturer and/or in the published literature. Note that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive, the data must show that the antigen being detected in the intended application — here, flow cytometry — is the desired target, using one or more of the approaches outlined below.

Knockout cell line approach

Genetic knockout-based validation can conclusively prove the ability of an antibody to detect the antigen of interest when expressed at endogenous levels (Laflamme et al., 2019). CRISPR-Cas9 approaches are commonly used to produce KO cell lines, some of which are commercially available. Various proteomic and transcriptomic datasets can be used to select a candidate cell line (e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen KO line are likely to express the target at a robust level, similar to that in samples of interest. If you are working with cells that are amenable to genetic knockout, this represents a very robust approach to antibody validation. If your protein of interest is essential to the cell’s function, it likely is not amenable to knockout approaches. In these cases, we would recommend a knockdown approach instead.

In this approach, you’re looking for an antibody that detects the target in the parental cell line, but not in the iso-genic knockout control. This can involve cell surface labeling and/or intracellular labeling (or both). It is important to test for the protocol you intend to use in your experiment. An antibody that demonstrates selectivity for cell surface labeling may become less selective if used in intracellular labeling because the intracellular compartment may contain cross-reacting antigens not present on the cell surface. The presentation of antigens can also be affected by cell fixation and permeabilization techniques — and so it may be necessary to try multiple techniques.

For example, we have found that some antibodies perform well in a PFA-saponin fixation and permeabilization protocol, but very poorly in a methanol fixation-permeabilization protocol. Although the expected location of the target is an important factor in deciding which protocol is likely to work, we have found that it is often best to determine this by testing them. For intracellular targets, we usually test 3 different fix/perms (methanol, PFA-saponin, PFA-triton). The fix/perm solutions can make a significant difference in the efficacy of your antibody. When compared, 4% PFA and 0.1% saponin; 4% PFA and 0.1% Triton X-100; and methanol, we found that the optimal perm/fix solution depended on the specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when looking to label an intracellular target.

Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a green or violet, fluorescent dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1% saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus 647 secondary antibodies. Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the KO population (pink histogram) compared to the WT cells (green histogram). The unfilled histograms show cells that have only been stained with secondary antibodies, showing the level of background staining. All antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody, ** = recombinant antibody. Figure adapted from Biddle et al., 2024.

Once an antibody is found that shows separation between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize the separation and reduce any background staining. This can include using: different blocking reagents; titration of antibody concentration (this can also save money!); and optimizing fixation/permeabilization reagents for intracellular targets.

Since knockout experiments can be time-consuming and expensive, it may be helpful to find an antibody that has already been validated for flow cytometry through this approach. If you’re looking for a flow antibody, the YCharOS (Antibody Characterization through Open Science) initiative has now started producing flow cytometry characterization data for selected antibodies, using the knockout approach. You can see an example of their work on their synaptotagmin 1 page. The data produced is available on the F1000 gateway and Zenodo community.