Research antibodies are critical for innovation and discovery, but the use of poorly characterized reagents can negatively impact the rigor and reproducibility of biomedical science. A new proposal published September 5 in Nature Methods shows the way to better research tools.
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 (IWGAV).
A few months ago in a separate post, I discussed the ongoing work by the IWGAV ─ an independent group of international scientists with shared goal of improving antibody validation. Thermo Fisher Scientific has provided financial support to the IWGAV since 2015 to lead the charge in addressing one of the biggest challenges associated with antibody specificity and reproducibility that has been plaguing biomedical researchers for decades.
On September 5, 2016, a report from our group entitled “A Proposal for Validation of Antibodies” was published online in the journal Nature Methods. [UHLEN 2016] This report provided, for the first time, a comprehensive proposal for scientific approaches to validate antibody specificity. We also developed recommendations for improving reproducibility in antibody use, which were published in the same report. In the publication, we proposed five key “conceptual pillars” for antibody validation based ontechnologies introduced by the genomics and proteomics communities: [UHLEN 2016]
- Genetic strategies: Antibody specificity is evaluated by comparing samples in which the intended target gene has been deleted or depleted with samples containing the functional gene expressed at normal levels
- Independent antibody strategies: Antibody specificity is evaluated by comparing detection patterns of a new antibody to an antibody with known properties
- Orthogonal strategies: Antibody specificity is determined by correlating the amount of antibody labeling to the expression level of the intended target protein across multiple samples. The expression level of the intended target protein is determined by an antibody-independent method (e.g., mass spectrometry [MS])
- Expression of tagged proteins: Antibody specificity is evaluated by comparing antibody labeling with expression of a tagged version of the target protein expressed from the endogenous locus. In many cases the tag will be a fluorescent protein that can be detected through direct observation
- Immunocapture followed by mass spectrometry (MS): Immunocapture is coupled with MS to detect proteins that bind the antibody being evaluated. This method may detect proteins that interact directly with the antibody, as well as those that form a complex with the directly bound target
Since that time, we have continued our efforts to engage the biomedical research community to gain feedback from and adopt a comprehensive standard for antibody use and validation. In late September, I, and other members of the IWGAV, participated in a meeting organized by the Global Biological Standards Institute (GBSI) held at the Asilomar Conference Grounds in California [SERVICK 2016]. The five conceptual pillars developed by the IWGAV formed the basis for discussion of application-specific standards for validating antibody specificity, which must be a core element of any comprehensive guidelines proposal developed by the GBSI [GBSI 2016]. The objective of the meeting was to provide a clear path toward specific, tangible and workable guidelines for antibody use and expanded our efforts by incorporating viewpoints from antibody producers, publishers, research funders, as well as wider group of antibody users.
Consensus from the meeting reiterated several recommendations from the IWGAV, including the need for application-specific and context specific validation of antibody specificity.
A comprehensive set of guidelines for antibody use will certainly help provide clarity that is desperately needed by antibody users, producers, publishers, and research funders. However, such guidelines must also be developed carefully to ensure recommendations are practical, workable, and usable by all stakeholders. While these deliberations take place, the report from the IWGAV provides an actionable set of recommendations that should be within reach of all stakeholders. As an example, a new version of the Human Protein Atlas will be launched on December 4 where the IWGAV recommendations have been applied to the existing data, and will be displayed on new antibody validation pages for every protein present in the database. With a concrete proposal in hand, and comprehensive guidelines on the horizon, the future looks bright for improvements in that antibody use and validation that will ultimately enhance the rigor and reproducibility of scientific research. However, with much left to be done, ultimate success will depend on active contributions from all stakeholders to the development of comprehensive guidelines for antibody use.
|BAKER 2015||Baker M. Nature. 2015 Nov 26;527(7579):545-51.|
|BORDEAUX 2010||Bordeaux J, et al. Biotechniques. 2015;521(7552):274-276.|
|GBSI 2016||Global Biological Standards Institute. Three Key Approaches to Increasing Research Antibody Validation Defined at GBSI Workshop Event Was Most Representative Gathering of Scientific Experts Ever Convened on the Topic. September 28, 2016. https://www.gbsi.org/press-release/three-key-approaches-increasing-research-antibody-validation-defined-gbsi-workshop-event-representative-gathering-scientific-experts-ever-convened-topic/. Accessed October 11, 2016.|
|SERVICK 2016||Servick K. Science. Validate your antibodies to improve reproducibility? Easier said than done. September 28, 2016. http://www.sciencemag.org/news/2016/09/validate-your-antibodies-improve-reproducibility-easier-said-done. Accessed October 11, 2016.|
|UHLEN 2016||Uhlen M, et al. Nat Methods. 2016 Oct;13(10):823-7.|