Genetic strategies for antibody validation

Researchers invest precious resources in studies that rely on the specificity of their chosen antibody. Often, the success or failure of a project hinges on the specificity of an antibody: whether it consistently recognises its target with little or no cross-reactivity. Clearly, researchers need to feel confident in the performance of such a key tool in their work.

It is important that antibody specificity is demonstrated in the particular technique in which the antibody will be used; an antibody that has been validated by western blot, for example, may perform very differently in immunocytochemistry.

Ideally, then, validation of a given antibody should be carried out across as many applications as possible. However, there are currently no widely accepted standards or practices for antibody validation.

In 2016, the International Working Group for Antibody Validation proposed five “pillars,” each representing a type of strategy for tackling antibody validation. The first of these covers genetic methods.

Genetic strategies for determining antibody specificity

Antibody specificity can be determined using cells or tissues that do not express the target protein of the antibody. In these cells, the target gene is “knocked out” or “knocked down” using genetic methods such as CRISPR-Cas9 or RNA interference (RNAi). These techniques can substantially reduce or eliminate the expression of the target protein. Any remaining signal detected using western blot analysis, for example, suggests cross-reactivity of the antibody. The knockout/knockdown cells can therefore act as powerful negative controls in antibody validation.

There are a number of techniques that can be used for gene editing as part of the antibody validation process, including:

CRISPR-Cas9

In this system, researchers create a piece of RNA with a short guiding sequence that binds to a specific DNA location in the target genome. The RNA also attaches to the Cas9 enzyme, which cleaves the DNA at the intended protein-coding sequence. Once the DNA has been cut, researchers can add, remove or change the gene as necessary. In this way, CRISPR-Cas9 technology can be used to develop “knockout” cells that do not express the target protein of a chosen antibody.

CRISPR-Cas9 is a popular choice among researchers because it is cheaper, quicker and more accurate than other methods of gene editing. However, one disadvantage of knockout methods is that, as they alter the DNA to prevent the target protein ever being expressed, there is a risk of cell death if that protein is essential for cell survival.

RNAi/siRNA

An alternative approach to removing or editing the target gene is to temporarily “silence” it. RNAi techniques recruit the cell’s own genetic machinery to “knock down” expression of the target protein.

One of the most common forms of RNAi involves siRNA (small, or short, interfering RNA). Composed of short, double-stranded pieces of RNA, siRNA can trigger the knockdown of a specific cellular mRNA sequence. The siRNA cuts the target mRNA at a particular site and the mRNA is then broken down by the cell. The result is a loss of protein expression.

In contrast to knockout techniques like CRISPR-Cas9, the loss of protein expression caused by siRNA is relatively short-term. Researchers have sometimes experienced problems with off-target effects of siRNA; however, a number of functional studies have helped to refine siRNA design and alleviate these concerns.

Genetic techniques for antibody validation have many advantages – but can also be expensive and resource-heavy. At St John’s Laboratory, we understand that carrying out such techniques in-house is simply beyond the capacity of many smaller research groups.

Our innovative Antibody Validation Project allows researchers to try up to five free, trial-size samples of our primary antibodies to assess their potential usefulness. With around 12,000 antibodies included in the project, we have a product to suit almost any requirement.

After running their own validation processes, researchers can return their results to us. These results are freely available on our website to anyone interested in our products, whether they choose to take part in the project or not. This gives researchers access to a wide range of antibody validation results from tests conducted in a variety of environments and, importantly, across very different applications.

Reference:

• Uhlen M. et al, A proposal for validation of antibodies. 2016. Nature America, Inc.