Specific antibodies need specific validation

One of the most utilized tools in biomedical research is the monoclonal antibody. These proteins have the potential to seek out and bind to any desired target, and can be used for cell imaging, cell sorting, immunoassays and many other applications.

But these lab workhorses don’t always run true. Depending on the nature of its target, an antibody might be inconsistent in certain tests — binding to the wrong target to give false positive results, for instance. Given the prevalence of research antibody use, this is potentially a billion-dollar problem.

A major goal is to develop antibody validation strategies so that researchers can have confidence that an antibody is suitable for their particular needs — and that their results will be reproducible.

Thermo Fisher Scientific has developed a two-part antibody validation platform to test not only the specificity of its Invitrogen^TM antibodies (that they bind to the right target), but also their suitability for different applications. However, the same test is not appropriate for all antibodies: Thermo Fisher uses an appropriate test for each protein target, depending on its biological function. Certain antibodies will be best tested using CRISPR-Cas9 to knock out the gene that encodes the target protein, and checking that the antibody no longer binds to anything. Other antibodies might be tested using immunoprecipitation followed by mass spectrometry to check that they are bound to the right targets.

Thermo Fisher is developing and refining antibody validation tests based on biological function of the target antigen. Here are two case studies of specific proteins and their specificity tests.

Knock-outs for cancer

The epidermal growth factor receptor (EGFR) is a well-studied protein: dysregulation in the EGFR pathway is implicated in various cancers. In order to test whether antibodies are specific to EGFR or to any of its downstream targets, researchers can knock out critical proteins in the EGFR pathway and see how the antibody-binding signal changes.

In recent years, the CRISPR-Cas9 system has become known as the most reliable and powerful way to knock out a gene. This makes it ideal for testing antibody specificity within a signalling cascade. Thermo Fisher researchers took a standard human carcinoma line (A-431) and used a western blot to get a baseline for the binding signal. They then used CRISPR-Cas9 to eliminate the target gene and create EGFR knockouts. A western blot of protein extracted from these knockout cells showed that there was no longer any signal for a target protein (Figure 1).

Further tests confirmed the result. The signalling cascade downstream of EGFR includes proteins such as RAS, RAF, MEK and ERK. Activation of EGFR by epidermal growth factor (EGF) leads to phosphorylation of these downstream proteins, which can be detected using other antibodies that recognize these phosphorylated states. However, adding EGF to the EGFR-knockout cells should not result in any downstream phosphorylation. Adding the same antibodies that recognize phosphorylated targets produced no signals. Thus, Thermo Fisher researchers are confident that the anti-EGFR antibody is target-specific.

An array of modifications

In the cell nucleus, DNA is tightly packaged — wrapped around histone proteins to form chromatin. Studying histones is difficult, as they can be affected by a number of chemical changes, known as post-translational modifications (PTMs). For example, residues on a histone can gain one or more methyl, acetyl or phosphoryl groups, which each have an effect on cellular function.

Certain techniques, such as chromatin immunoprecipitation (ChIP), western blotting, immunofluorescence and immunohistochemistry, use antibodies against specific histone PTMs to understand the state of the histone and its binding. However, several histone modifications have similar DNA-binding patterns; an antibody that has not been rigorously tested against all histone PTMs might bind to the wrong type and deliver a false-positive result.

Thermo Fisher tested its histone PTM-specific antibodies using an array of peptides bearing a variety of PTMs. If an antibody is truly specific to one PTM, it will bind only to those spots that carry that PTM. Thermo Fisher researchers measured the signals using a specificity factor: the average intensity of all spots containing a particular PTM divided by the average intensity of all spots without it (Figure 2). The antibodies showed a 4- to 190-fold higher specificity factor for their target PTM state than non-target states, giving confidence that they are highly selective.

Thermo Fisher has seven other specificity tests beyond genetic knock out and peptide arrays. These include using RNAi to knock down gene expression, a differentially raised antibody to independently verify targeting, and naturally occurring variable expression to confirm specificity. Only through such careful and rigorous testing can researchers be confident that their lab workhorses are fit for purpose — and that their work will stand up to the closest scrutiny.

Find application notes on these Invitrogen antibodies and more about Thermo Fisher Scientific’s two-part testing approach here.