The Foundations of Antibody Validation

For many life science researchers, antibodies are an essential part of the laboratory toolkit. They are the foundation of many important scientific discoveries, as well as the building blocks of innumerable publications. Antibodies are most commonly employed in western blot assays (immunoblotting), and are also regularly used in immunohistochemistry and immunocytochemistry.

Whatever the intended purpose, researchers need to be confident in their chosen antibody. The antibody validation process seeks to provide this reassurance by demonstrating that a given antibody is specific, selective and reproducible in a particular context. When carried out to a high standard, antibody validation shows researchers that their chosen antibody will behave in the way they expect.

Researchers seeking reassurance are often disappointed, however, as there is currently no widely used framework for antibody validation across different applications. An antibody that behaves impeccably in one method may prove unpredictable in others, yielding unexpected or inconsistent results. Careful study of the behaviour of an antibody in western blot assays may be of little relevance if the antibody is to be used in ELISA. It is, therefore, vital that antibody validation is carried out for specific applications.

In 2016, the International Working Group for Antibody Validation was convened to tackle this problem. The group proposed five strategies, or “pillars”, to be used as guidelines for robust validation. Those five pillars are:

Genetic strategies

These techniques measure antibody specificity by first using methods such as CRISPR-Cas9 to knock out or knock down the target gene in control cells. The expression of the target protein is eliminated or substantially reduced, so any remaining signal suggests cross-reactivity of the antibody.

Orthogonal strategies

This approach involves using non-antibody methods to quantify the target protein, then comparing the results with those achieved using the antibody-based method. Researchers using this strategy should try to examine a large number of samples that vary in their expression of the target protein. This increases confidence in the specificity of the antibody and allows for robust statistical analysis.

Independent antibody strategies

These strategies utilise two or more antibodies that bind to different regions of the same target protein (and are therefore “independent” antibodies). Correlation between the expression patterns produced by the different antibodies in a particular environment suggests antibody specificity.

Expression of tagged proteins

As the name suggests, antibodies can be validated using the expression of either a protein containing an affinity tag or a fluorescent protein. For the former, parallel detection with validated immunoreagents, and for the latter, direct observation, can show specificity and selectivity off the antibody under investigation. A difference between the patterns generated by the chosen antibody and the tag or fluorescent signal suggests cross-reactivity.

Immunocapture followed by mass spectrometry

A chosen antibody can be used to isolate a protein from a solution: this is immunocapture. When combined with mass spectrometry, this strategy can identify proteins that will bind to the antibody, as well as proteins that may interact indirectly with the target protein.

Ideally, an antibody should be validated using a combination of these five strategies. The broader the validation, the more confidence researchers can have in their chosen antibody – and therefore also in their investment of precious time and money.

At St John’s Laboratory, we understand the need for robust antibody validation. Our innovative Antibody Validation Project gives all our customers the opportunity to try up to five free, trial-size samples of our primary antibodies. Customers who report their own validation results to us can receive a discount on purchases. This not only allows customers to try a range of samples before making their final selections, but also helps us to build a database of real, up-to-date validation results as a resource for the wider research community.

All our validation data is freely available on our website.