Antibody Validation for Flow Cytometry

Validation provides proof that an antibody both recognizes its specific target and performs in a given application. For flow cytometry, a technique that often involves detecting multiple targets simultaneously, it is essential to have confidence that antibody reagents are behaving reliably. In this article, four respected antibody manufacturers explain how they validate antibodies for flow cytometry and share some tips for antibody selection.

Validation is critical to produce accurate data

According to Rob MacDonald, Scientist in the Flow Cytometry Group at Cell Signaling Technology, the goal of antibody validation is to be able to demonstrate that an antibody binds strongly to the intended target and does not bind other targets. “Antibody reactivity should be established on a species-by-species basis, barring instances in which a target shares 100% sequence identity with a validated species,” he says. “Additionally, antibodies should be validated for every application in which they will be used, with each validation process adhering to a well-defined and reproducible protocol.” Importantly, validation should aim to reflect the performance of the antibody in alignment with known biology; this ensures an accurate readout and enables unknown biology to be explored. Once an antibody manufacturer has completed validation, the onus is then on the end user to verify antibody performance in their own particular setting.

Challenges of validating antibodies for flow cytometry

When validating antibodies for flow cytometry, the degree of difficulty often depends on the target location. “Validating antibodies for use on live cells can be straightforward since the potential for off-target binding is limited by the plasma membrane, which prevents antibodies from accessing intracellular targets,” explains MacDonald. “However, validating antibodies against intracellular targets is often more challenging because flow cytometry does not provide high-resolution subcellular localization information or insights into molecular weight.” Moreover, while some antibodies work well on live cells, they may not perform after cells have been fixed and/or permeabilized; establishing this during the validation process is key.

Another challenge associated with validating antibodies for flow cytometry lies in identifying appropriate model systems to determine antibody specificity. Unlike techniques such as Western blot, which rely on samples pooled from many different cell types, flow cytometry provides analysis of distinct cellular populations, meaning selecting appropriate cell types for validation purposes is key. “Validation begins with understanding the biology,” reports Kenta Yamamoto, Product Manager for Cell Analysis at BioLegend. “We typically seek this information from published literature citing expression of a particular target, as well as looking at other clones that are widely used in the field. Multicolor flow cytometry then allows us to detect co-expressing and mutually exclusive markers to determine whether the antibodies we develop are binding the expected cell populations.”

The multiplexed nature of flow cytometry brings its own problems. Dr. Justyna Zaborowska, Product Manager in Cancer at Bio-Rad, notes that validating antibodies for flow cytometry typically involves using a broader range of controls than are required for many other immunoassay techniques. “Common flow cytometry controls include unstained cells, isotype controls, secondary antibody only controls, dual stains, and positive and negative controls,” she says. “Each of these provides valuable information about antibody performance to help ensure the correct target is specifically recognized.” Critically, once antibodies have been validated individually, their performance in the intended multiplex panel must also be assessed.

Image: Confirmation of IL2 staining in stimulated CD3 positive cells versus matched isotype control and unstimulated control. A) Unstimulated cells were stained with Alexa Fluor 700 conjugated Mouse anti Human CD3 (MCA463A700) and FITC conjugated Rat anti Human IL-2 (MCA1553F). B) Cells stimulated with Cell Stimulation Reagent containing Brefeldin A (BUF077A) for 5 hours were stained with Alexa Fluor 700 conjugated Mouse anti Human CD3 (MCA463A700) and FITC conjugated rat IgG2a (MCA6005F). C) Cells stimulated with Cell Stimulation Reagent containing Brefeldin A (BUF077A) for 5 hours were stained with Alexa Fluor 700 conjugated Mouse anti Human CD3 (MCA463A700) and FITC conjugated Mouse anti Human IL-2 (MCA1553F). All experiments performed on red cell lysed human blood gated on lymphoid cells in the presence of 10% human serum. Data acquired on the ZE5 Cell Analyzer. Image provided by Bio-Rad.

Validation is multi-faceted

“Validation is far from being a one-step process,” cautions Sreethu Sankar, Research and Development Manager at Proteintech Group. “Instead, it encompasses everything from antigen design and clone selection through to evaluation of antibody performance in the chosen application.” He also notes that an application-focused antibody development approach can significantly improve the specificity and sensitivity of the final product, suggesting as an example that flow cytometry-based clone selection (rather than ELISA) should be used when developing antibodies for flow. “Whether you are developing antibodies to a conventional immunology target such as a CD marker, or to an intracellular protein such as a cytokine or transcription factor, you cannot expect an antibody designed for ELISA to work in flow cytometry due to the difference in epitope conformation,” he says. Despite this, many antibodies do work in multiple applications, but antibody performance should always be confirmed on a case-by-case basis.

“Where antibodies are useable in other applications, testing via various techniques helps further verify specificity,” notes Yamamoto. “For example, flow cytometry and Western blot each potentially address different binding properties of an antibody, being used to detect native and denatured proteins, respectively.” The more antibody data that is available, the greater the body of evidence that an antibody is performing as it should. “Applications that follow similar protocols can especially be helpful for validation,” adds MacDonald. “Take immunofluorescence microscopy, for instance, which can alert researchers to false positives generated during flow cytometry-based validation by enabling any improper subcellular localization to be seen.” Using non-antibody-based techniques (orthogonal validation) can also help support antibody-based findings.

Tips for antibody selection

The first step toward generating reliable flow cytometry data is to source antibody reagents from a trusted supplier. “Researchers should always review the data generated by the manufacturer to confirm that an antibody has been validated for flow cytometry, in the right cell model, with all the required controls,” says Sankar. Next, researchers should follow the antibody manufacturer’s recommended protocol to safeguard antibody performance. Notably, with complex cellular models such as human peripheral blood and mouse tissues seeing frequent use in research, MacDonald reports that Cell Signaling Technology validates many of its antibodies by flow cytometry in these systems and can share protocol advice on request.

“When selecting clones that have not been widely published, check with the antibody manufacturer to determine exactly what types of validation studies have been performed,” advises Yamamoto. “When we advertise an antibody as being raised against a particular antigen, we try to address any potential off-target effects as thoroughly as possible. We are always happy to share this information with researchers to help them decide whether a clone suits their needs.”

Lastly, Zaborowska highlights the guidelines proposed by the International Working Group on Antibody Validation (IWGAV) and published in Nature Methods, which provide a framework for antibody validation across different research applications. “By aligning our validation methods with the five pillars proposed by IWGAV, we aim to help researchers produce the highest quality data,” she says. “For instance, we regularly use CRISPR/Cas9-mediated gene knockout, siRNA-mediated knockdown, and immunoprecipitation followed by mass spectrometry (IP/MS) to confirm antibody specificity. Although validation can be both costly and time-consuming, it is essential to improve the value and reproducibility of published results.”