Antibody Validation Methods

Drawing accurate conclusions from immunoassay data means having confidence in the antibodies that you use. But, with millions of antibodies available on the market—some of better quality than others—finding the right product for your research can be a difficult task. This article comments on what makes a well-validated antibody and shares tips for verifying antibody performance in your own model system.

Identifying potential antibodies

Carefully scrutinizing antibodies before making a purchase is a critical step in obtaining a product that meets your needs. Biocompare’s search functionality allows you to quickly identify multiple vendors for an antibody of interest, but you should then examine the associated product datasheets, reviews, and citations to home in on a suitable antibody for your intended use. “A vendor’s datasheet should contain clear, detailed information covering both antibody production and performance,” reports Antonella Galli, Ph.D., Head of Characterization at Abcam. “Key manufacturing specifics comprise the name of the target protein, clone number, host species, isotype, purification method, formulation, and quantity. Performance specifics should include convincing validation data confirming the antibody’s successful use in particular applications, along with details of how those data were generated.” Where product citations are used to guide antibody selection, Galli cautions adopting a critical approach, especially for novel targets where data may be scarce or conflictual. “In these instances, using a reliable vendor that can provide validation data across applications is recommended to ensure the antibody you receive is specific, high-affinity, sensitive, and reproducible,” she says.

Seeing data from multiple validation strategies is essential

So, what makes convincing validation data? Emily Cartwright, Senior Product Marketing Specialist for Antibodies at Bio-Techne, explains that where validation involves using multiple strategies, it provides a more complete picture of how an antibody will perform. “At Bio-Techne, we validate our antibodies for specificity based on the 5-Pillar Model from the International Working Group for Antibody Validation,” she says. “This comprises genetic strategies such as knockout and knockdown; biological or chemical modulation of protein expression; using an independent antibody targeting a different epitope; using a tagged or overexpressed protein; and non-antibody based, orthogonal methods such as RNAscope^® ISH technology, which detects RNA and can be combined with protein detection by antibodies in the same cell. By sharing details of these tests on our product datasheets, we offer visual proof of antibody specificity, as well as provide a useful starting point from which researchers can perform similar validation experiments in their own model systems.” 

Bio-Techne’s LC3B antibody, NBP2-46892, is validated using both knockout (KO) and biological strategies. Western blot comparison of wild-type and LC3B KO cell lines, either untreated or treated with chloroquine (validation by chemical modulation), shows no LC3B expression in the KO cell line and increased expression in chloroquine-treated wild-type cells.

Also highlighting the value of employing multiple strategies for antibody validation, Joanna Porankiewicz-Asplund, Ph.D., Technical Support Manager at Agrisera, comments that direct and indirect validation are distinct. “For antibodies to targets that are vital to cellular function, such as photosynthetic proteins, obtaining viable mutants is not possible,” she says. “Consequently, in this situation, indirect validation methods must be used. These include checking that the antibody detects a band of the correct molecular weight on a Western blot—taking any processing or modifications into account—and confirming that the same band is not present when the membrane is incubated with pre-immune sera; determining whether a recombinant form of the protein is recognized; performing competition studies with a free peptide; running tests with samples from specific conditions known to up- or downregulate target protein expression; and assessing cross-reactivities.” She also advises that selecting antibodies supplied only with a titer guarantee in indirect ELISA (where the microplate wells are coated with the immunizing protein or peptide) may not be a sensible choice. “On its own, indirect ELISA is not enough to confirm future reactivity of a given antibody in endogenous sample material,” she says.

According to Eileen Gonzalez, Ph.D., Associate Product Manager for Antibody Applications at BioLegend, seeing application-specific validation data is essential. “Despite being raised against the same antigen targets, antibodies do not always perform with the same efficiency across different assay formats,” she explains. “For this reason, it is important to source antibodies that are proven to work in your chosen application and that have, ideally, been validated with the same tissues or cells as your sample type. A good manufacturer will have completed testing across samples exhibiting varying levels of endogenous target expression and will have run knockout controls whenever possible. When a product that has been validated for your chosen application and/or sample type does not appear to be available, a manufacturer offering personalized support may be able to provide further guidance. This could include suggesting whether an antibody is likely to work in a particular application or if testing in additional sample types is currently underway.”

Sustained effort by antibody manufacturers

With researchers increasingly questioning antibody quality, manufacturers are working hard to enhance their validation processes. “At Abcam, we validate our antibodies as closely as possible to our customers’ needs so that they can easily use them in their own model systems,” reports Galli. “This includes performing validation studies in primary cells and tissue microarrays, and we also recently launched a customer-centric antibody validation package for key immuno-oncology recombinant antibodies, which we believe will be of particular interest to biopharma customers.”

Agrisera has a strong focus on improving antibody validation for plant and algal cell biology, where a vast array of species, tissues, and sample types is being investigated. “Having access to suitable material—from organs like leaf, root, algal, or diatom cells—and available mutants is essential for properly validating an antibody,” explains Porankiewicz-Asplund. “Through collaboration with laboratories worldwide, we have been able to use the most relevant sample types for validating antibodies to plant and algal proteins which, in turn, has enabled us to develop methods for improved chances of target detection. For example, highly specific extraction buffers are required for certain target proteins, which may also require that isolation be carried out under blue light or using a particular extraction technique.”

Validation of a PsbA antibody using extracts from different plant species. 1) Arabidopsis thaliana, 2) Hordeum vulgare, 3) Chlamydomonas reinhardtii, 4) Synechococcus sp. 7942, 5) Anabaena sp. Image provided by Agrisera.

BioLegend regularly employs additional testing methods alongside its existing validation strategies. “Often, we will adapt the types of samples we use for validation to better align with what is known about the target,” reports Gonzalez. “For instance, if it has been shown that murine kidney tissue has 10-fold higher expression of a given protein compared to brain tissue, it is common for our product development teams to check the antibody specificity against both tissue types. We also frequently generate knockout cells on demand, using RNAi, shRNA, or CRISPR-Cas9 technologies.”

Lastly, Cartwright notes that to aid researchers in performing their own validation, Bio-Techne offers a growing number of products to support these types of independent studies. “Our off-the-shelf products for externally validating antibodies include cell lysates, tissue slides, and RNAscope^® probes and kits,” she says. “Moreover, we continually strive to develop high-quality antibodies and increase the range of products available for their validation—not only for use by our in-house teams but also by our customers.”