The Path to Antibody Specificity Confirmation

Editor’s note: research antibodies are critical for innovation and discovery, but the use of incompletely characterized reagents can negatively impact the rigor and reproducibility of biomedical science, potentially leading to wasted time and resources. Comprehensive antibody validation standards and specificity confirmation will help researchers proceed with confidence, knowing they can count on the reagents they use.

About Mathias Uhlén: Dr. Uhlén, Professor of Microbiology at Royal Institute of Technology, Sweden, is a noted protein biochemist, the founder of the Human Protein Atlas, and Chair of the International Working Group on Antibody Validation.

Research antibodies are one of the workhorses that drive innovation and discovery across the biomedical sciences: on average, a typical scientist may perform more than three antibody-based assays each week. [BIOCOMPARE 2015] Frequent use of antibodies in the laboratory highlights the fundamental importance of antibody-based techniques for modern science, from the study of stem cells to investigation of cancer biology. Frequent antibody use also illustrates how the systematic application of incompletely characterized antibodies (and misuse of those that are well characterized) might quickly lead to confusion, misinterpretation of data, and irreproducibility in the scientific literature. These concerns (first ignited in 2012 by reports that only a small proportion of landmark pre-clinical studies in oncology could be reproduced by scientists at Amgen) have led to calls by the National Institutes for Health (NIH) and others to standardize the use of research reagents, including antibodies. [BAKER 2015B; BEGLEY 2012; COLLINS 2014]

Recognition of potential pitfalls with antibody use is nothing new. As early as 2009, a study of 24 antibodies thought to specifically recognize different muscarinic receptor (MR) subtypes found that each antibody (with only 2 exceptions) recognized additional proteins expressed in tissues of corresponding MR-receptor-knockout mice. This finding led the authors to conclude that “reliable immunohistochemical localization of MR subtypes with antibodies is the exception rather than the rule.” [JOSITCH 2009]  Published reports like this one (and likely, the personal experience of countless other investigators) have led to calls for universal standards to improve antibody specificity and reproducibility to ultimately enable more effective biomedical research. (Figure 2) [FASEB 2016; BORDEAUX 2010; BAKER 2015B]

 

 

Given the critical need for expanded antibody validation standards, including for antibody specificity confirmation, developing the underlying strategies used to confirm antibody specificity and improve reproducibility poses the next challenge. I would argue that the most successful approaches will adhere to four key attributes:

•         Validation strategies should account for differences across key research applications in which antibodies are used:  Research antibodies can be generated in a number ways, and may function differently across applications.  For this reason, recommendations must be made with each of the key applications in mind. Adequate reporting of application-specific protocols and data will also be critical to ensure reproducibility among all users
•         Validation strategies should encourage adoption of the latest technology: Although the potential pitfalls with antibodies are nothing new, the ability to characterize antibodies, particularly in terms of their specificity, has changed dramatically.  Emerging technologies developed by the genetics, genomics, and proteomics communities, should be leveraged in validation assays to provide the strongest evidence an antibody recognizes its intended target
•         Validation strategies should be developed with both providers and users in mind: Discussions surrounding antibody validation often focus only on antibodies themselves; just as important are the ways in which antibodies are put to use by scientists in the laboratory. In addition to providing recommendations for validation of antibody specificity, proposals should recommend approaches for transparency in the use of antibodies and reporting of antibody-derived data
•         Validation strategies should incorporate community input: For widespread acceptance and adoption, validation strategies will need to account for the diversity of views across the communities who will use them.  For this reason, community input should be integrated to ensure any proposal will meet the needs of researchers at the bench.  Input from a wider group of stakeholders including publishers, research funding agencies, and antibody providers should also be encouraged

Developing a proposal for antibody validation strategies along these lines is precisely the approach being taken by the International Working Group on Antibody Validation (IWGAV), of which I am the chair. At our inaugural meeting last September, we set forth a plan to develop best-practice approaches to validate antibody specificity in the most commonly-used research applications. We also sought to provide recommendations to improve reproducibility for both antibody providers and users.

Since then, our group has met regularly, with operational support from Thermo Fisher Scientific, and is committed to the goal of releasing a proposal within one year of the group’s inception. Ultimately, we hope that adoption of these prospective validation principles will result in greater confidence among antibody users and more reliable and reproducible research outcomes.

 

References:

BAKER 2015A	Baker M. Nature. 2015 May 21;521(7552):274-6.
BAKER 2015B	Baker M. Nature. 2015 Nov 26;527(7579):545-51.
BIOCOMPARE 2015	Biocompare. Antibody Market Report 2015. http://www.biocompare.com/Editorial-Articles/177815-2015-Antibody-Market-Report/. Published September 8, 2015. Accessed June 12, 2016
BORDEAUX 2010	Bordeaux J et al. Biotechniques. 2015;521(7552):274-276.
BEGLEY 2012	Begley CG, Ellis LM.Nature. 2012 Mar 28;483(7391):531-3.
BERGLUND 2008	Berglund L et al. Mol Cell Proteomics. 2008;7(10):2019-2027.
FASEB 2016	Federation of American Societies for Experimental Biology. Enhancing research reproducibility: recommendations from the Federation of American Societies for Experimental Biology.  http://faseb.org/Portals/2/PDFs/opa/2016/FASEB_Enhancing%20Research%20Reproducibility.pdf Published January 14, 2016.  Accessed 6/13/2016.
JOSITCH 2009	Jositsch G et al. Naunyn Schmiederbergs Arch Pharmacol. 2009;379(4):389-395