Antibodies are proteins that bind with high affinity and specificity to target molecules. They are one of the most used tools for studying protein biology. Some have been produced to recognize proteins of interest, while others recognize short peptide chains (peptide tags) that are bound to proteins to enable detection or isolation.
The ability of research antibodies to bind to their proteins of interest or peptide tags, varies depending on the amino acids which surround the target binding sites. However, according to recent studies published in Science Signaling, the authors have warned that in many cases, such antibodies do not bind as expected.
Insufficient validation of antibodies?
Pathologist David Rimm from Yale School of Medicine who was not part of the research team that published the new studies, told The Scientist recently:
“This issue keeps popping up its head because people don’t follow directions for validation. [Researchers] don’t take it seriously enough,” he says, explaining that there is still data from experiments with unvalidated antibodies being published in the literature.
The claims in the recent studies have been repeated by pharmacologist Thomas Wieland of Heidelberg University: “These papers, like others, clearly indicate that even commercially available antibodies need thorough evaluations for the respective purposes for which they will be used.”
Egon Ogris (Medical University of Vienna), who authored the two studies, is similarly frustrated. His concerns first arose after working with a specific commercial antibody that is commonly used, he felt compelled to examine the issue in more depth, as a “service to the community,” he says.
People need to know the danger attached to antibody use, and if you do not want to be tricked by your antibodies, then you need to validate them.
― Egon Ogris, Medical University of Vienna
Ogris’ group studies the ubiquitously expressed multi-subunit enzyme, phosphatase 2 (PP2A), which is involved in numerous cellular functions and implicated in diseases including cancer and neurodegeneration. To examine one of the subunits which forms the enzyme, he and a co-worker added Myc peptide tag to it. However, because they happened to use different enzymes to insert the Myc tags, they developed two versions of the tagged subunit that differed by just a few amino acids at the region which linked the tag to the protein.
When Orgis et al. chose to bind 9E10, a commercially available Myc-targeting antibody, to detect the subtly different tagged PP2A subunits they were greatly surprised by the results. One version of the protein produced a strong clear signal “and the other was hardly visible”.
“The only difference was these four or five amino acids,” Ogris says, suggesting this was the cause of 9E10’s failure to recognize the tag. In order to learn more, Orgis’ team produced their own Myc antibody, A46, and showed that unlike 9E10 their antibody could bind equally well to both tags and produce similar strength signals in both samples. Orgis’ team went on to examine four other commercially available Myc antibodies, subsequently finding that all but one exhibited variable binding affinity for the Myc tag depending on the surrounding amino acid sequences.
In their second study, Ogris et al. examined antibodies that directly target the catalytic subunit of PP2A. Many of these antibodies exist, but there are concerns that methylation of the subunit, a necessary modification for the enzyme to function, interfered with the antibodies’ binding abilities. Of the four antibodies tested, all but one showed a dramatically reduced affinity for the methylated version of the protein. Furthermore, the same three antibodies expressed a weak affinity for the related yet entirely separate phosphatase PP4.
All the tested antibodies are commercially available and one (1D6) forms part of a widely used PP2A activity assay kit. Because of his findings, Ogris advises other PP2A researchers not to use the kit.
A new method to validate antibodies?
There may be a light at the end of the tunnel for those looking to validate their antibodies thanks to emerging technologies based on microfluidic systems. One example is the Fluidity One-W which measures the binding affinity between proteins as well as reports on stoichiometry of complexes. By measuring protein interactions, fully in solution, the Fluidity One-W could give researchers a vital tool in antibody validation. Scientists such as Ogris are now strongly advising researchers to validate their own antibodies and not to expect commercial vendors to send them ones that have been rigorously tested. This is the only way that scientists can expect to yield expected reproducible results.
For more information on how you can validate the size and binding affinity of your antibodies click here