Do You Know if Your Antibodies were Validated?

Increase productivity immediately! Get the results faster! Get more funding now! Publish first! These are just a few of the internal and external time pressures that many scientists face every day regardless of geography or lab setting. To meet these demands, a scientist may find he or she must order critical experimental laboratory reagents from vendors promising quick delivery. The reagent arrives quickly, as promised, and the scientist conducts the experiment, presuming that the vendor and/or manufacturer has spent the time and energy needed to test the quality of the reagent and its usefulness for the particular experiment type being done. As it turns out, this is an often wrong presumption to make, resulting in multiple bad outcomes.

The US spends close to $800 million dollars a year on conducting research that use protein-binding reagents. A whopping $350 million of it is wasted due to bad reagents, particularly antibodies that fail to perform as expected (Bradbury & Plückthun, 2015). These are significant wasted resources that could have been used to further the scientist’s research more productively. So, how can the “faulty antibodies” be avoided in the first place?

The simplest answer is to validate the antibodies. But who should validate, and to what degree?

It seems obvious to state that the manufacturer should invest the time and resources to fully validate their antibody products before making them available. Actually, this may not be feasible because the sheer volume of antibodies that would need to be tested in numerous different assay formats that have unlimited number of buffers/conditions. Nevertheless, several companies are taking up the torch to validate some of their antibody products (Baker, 2015a).

Efforts have been made to establish third party groups to help validate antibodies. Several websites and a few antibody companies are gathering/sharing reviews, data or articles using particular antibodies, such as the Antibodypedia, Antibody Validation Channel, Biocompare, St. John’s Laboratory, and CiteAb (Baker, 2015a; Freedman et al., 2016). Some companies, including ThermoFisher Scientific and Abgent, are even offering to validate an antibody for the wary scientist.

But ultimately, the burden of responsibility for validating an antibody for a particular use or uses falls on the end user. According to a survey put out by the Research Antibodies and Standards Task Force (set up by the Global Biological Standards Institute), the vast majority of seasoned researchers (~6 or more years post-training) realize that this is the case (Freedman et al., 2016). But the same survey also revealed that less than 45% of researchers who recently completed their training take the time to validate their antibodies (Freedman et al., 2016).

What would prevent a scientist from making sure the antibody is good and the results are trustworthy? The very same survey revealed that time, money, and delay in research were the primary reasons why a scientist may elect to not validate an antibody (Freedman et al., 2016). Here’s the head-scratcher: If the antibody yields a false-positive result or unreproducible result, the researcher would have already wasted money, time, and experienced a huge set back in their research. It would appear that more seasoned researchers have learned this lesson, but it will take time for those with less experience to come to this realization.

What if the researchers who recently completed their training do not fully know how to validate their antibodies, but want to? Yale University’s David Rimm (having falling victim to dubious antibody performance and now a champion for antibody validation) developed a flowchart of methods that can be used for validating an antibody, including the use of cell-lines that have the expression of the antigen knocked down (Baker, 2015b; Bordeaux et al., 2010). The scientists could also perform labor-intensive pull-downs followed by mass spectrometry to identify the protein(s) that bound to the antibody.

Why are antibodies so difficult? Like life, antibodies can be complicated. They are created in either living animals or in cell-lines, which can lead to variations in antibody composition, post-translational modifications (chemical changes made after the antibody or protein is created) or complete loss of the product if a cell-line dies/fails to grow. This can cause batch-to-batch antibody variability or cause a product to become unavailable.

Aside from variations due to the origination from animals or cells, other factors can affect antibody performance. For example, the purification of the antibodies from animal blood or from cell-lines can vary in quality and in the amount of contaminating proteins. Improper shipping, handling, or storage (wrong conditions or using past the expiration date) of the antibodies may cause them to unfold and lose activity. Another source of performance problems can be attributed to the antigen used in antibody development, which may possess a different set of post-translational modifications compared to its counterpart found in tissue or other biological sample, thus affecting antibody binding. If the experimental conditions are different from those used to create the antibody, the antigen’s antibody binding-site could become obscured by either the antigen adopting a different conformation or the antigen forming different complexes with other biologics. Yet, another source of performance issues could be attributed to the antibody’s specificity: The antibodies may bind to proteins that are similar to the intended target or to extremely abundant proteins. These are by no means the complete set of reasons for the problems seen with antibodies in research (for a more complete list, see a recent review by Michael Weller (Weller, 2016)).

Clearly, antibodies can be variable not only in their composition, but also in their performance. It would be ideal if a scientist could use affinity reagents that were less prone to variability, such as SomaLogic’s SOMAmer® reagents. SOMAmers, which are made of chemically synthesized modified single-stranded DNAs, can be used in most laboratory assays in place of antibodies. Their chemical origin greatly reduces batch-to-batch variability and the other issues that arise when using antibodies derived from animals or cell-lines. The methodology used to create SOMAmer reagents also includes measures to improve the specificity and enhance affinity. The methodology can be adjusted to generate SOMAmer reagents better suited for binding the desired target in the experimental conditions. SomaLogic researchers characterize what each SOMAmer reagent binds to by using mass spectrometry for high abundance targets, pull downs, and binding assays using very similar proteins to check for specificity. Although this level of characterization gives the user some confidence, it is still up to the researcher to confirm that the specific SOMAmer reagent will work for their specific need.

The old saying “slow and steady wins the race” can apply when it comes to research. Time and money should always be invested to validate binding reagents – or other critical assay components – that will be used for an intended experiment. The external/internal pressures may never go away, but at least fewer resources will have been wasted and more meaningful and reproducible research can happen.

References

Baker, M. (2015a). Antibody anarchy: A call to order. Nature, 527(7579), 545-551. doi:10.1038/527545a

Baker, M. (2015b). Reproducibility crisis: Blame it on the antibodies. Nature, 521(7552), 274-276. doi:10.1038/521274a

Bordeaux, J., Welsh, A., Agarwal, S., Killiam, E., Baquero, M., Hanna, J., . . . Rimm, D. (2010). Antibody validation. Biotechniques, 48(3), 197-209. doi:10.2144/000113382

Bradbury, A., & Plückthun, A. (2015). Reproducibility: Standardize antibodies used in research. Nature, 518(7537), 27-29. doi:10.1038/518027a

Freedman, L. P., Gibson, M. C., Bradbury, A. R., Buchberg, A. M., Davis, D., Dolled-Filhart, M. P., . . . Rimm, D. L. (2016). [Letter to the Editor] The need for improved education and training in research antibody usage and validation practices. Biotechniques, 61(1), 16-18. doi:10.2144/000114431

Weller, M. G. (2016). Quality Issues of Research Antibodies. Anal Chem Insights, 11, 21-27. doi:10.4137/ACI.S31614