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COMMENTARY
A proposal for validation of antibodies
2016Nature America, Inc. All rights reserved.
2016Nature America, Inc. All rights reserved.
Mathias Uhlen1, Anita Bandrowski2, Steven Carr3, Aled Edwards4, Jan Ellenberg5, Emma Lundberg1, David L Rimm6, Henry Rodriguez7, Tara Hiltke7, Michael Snyder8 & Tadashi Yamamoto9
We convened an ad hoc International Working Group for Antibody Validation in order to formulate the best approaches for validating antibodies used in common research applications and to provide guidelines that ensure antibody reproducibility. We recommend five conceptual pillars for antibody validation to be used in an application-specific manner.
Antibodies are among the most frequently used tools in basic science research and in clinical assays. Despite their widespread use, as well as extensive and valuable discourse in the literature16, a comprehensive scientific framework for antibody validation across research applications is lacking. As a result, the quality and consistency of data generated through the use of antibodies vary greatly. This poses an impediment to the rigor and reproducibility that are the cornerstones of the advancement of science.
The extensive discussion of antibody validation in the literature indicates a collective need for standards to validate antibody specificity and reproducibility, as well as a need for adequate reporting practices. For example, in 2010, Bourbeillon and colleagues4 introduced the minimum information about a protein affinity reagent (MIAPAR) proposal. This proposal was meant to formalize a standard for how to report information about affinity binder
reagents so that the correct reagent for a particular target could be selected for a specific application. The MIAPAR proposal is a useful guide for this purpose; however, it does not include explicit recommendations for the experimental approaches best suited to support validation of antibody specificity in particular applications.
Immunoreagents are used in a range of applications. According to the antibody reagent portal Antibodypedia (http://www.antibodypedia.com
Web End =http:// http://www.antibodypedia.com
Web End =www.antibodypedia.com ; Supplementary Fig. 1), their most common application is in western blot assays (immunoblotting), followed by immunohistochemistry and immunocytochemistry. In addition, the sandwich assay (e.g., ELISA), although it encompasses only a low percentage of overall antibody use, is an important application from a clinical perspective.
It is essential to note that samples are treated substantially differently in preparation for different antibody-based assays (Supplementary Table 1). Proteins are typically in near-native...