Although poorly characterized antibodies are blamed for the loss of hundreds of millions of dollars—the bitter fruit of failed or unreliable experiments—dissatisfaction with antibody validation has yet to be adequately addressed. A comprehensive framework for antibody validation across research applications is still lacking.
In hopes of stimulating the research community to achieve a higher standard of antibody reproducibility, the International Working Group on Antibody Validation (IWGAV), an independent group of international scientists, issued a set of guidelines. These guidelines, which appeared September 5 in Nature Methods, emphasize that antibody validation is best pursued in an application- and context-specific manner.
In Nature Methods, representatives of the IWGAV, including the group’s chair, Mathias Uhlén, professor of microbiology at Sweden’s Royal Institute of Technology, published a paper entitled, “A Proposal for Validation of Antibodies.” The paper outlined five “conceptual pillars” to guide antibody validation in specific research applications:
- Genetic strategies: Measure the relevant signal in control cells or tissues in which the target gene has been knocked out or knocked down using techniques such as CRISPR/Cas or RNA interference (RNAi).
- Orthogonal strategies: Use an antibody-independent method for quantification across multitudes of samples and then examine the correlation between the antibody-based and antibody-independent quantifications.
- Independent antibody strategies: Use two or more independent antibodies that recognize different epitopes on the target protein and confirm specificity via comparative and quantitative analyses.
- Expression of tagged proteins: Modify the endogenous target gene to add sequences for an affinity tag or a fluorescent protein. The signal from the tagged protein can be correlated with detection through antibody-based methods.
- Immunocapture followed by mass spectrometry (MS): Couple immunocapture, the technique of isolating a protein from a solution through binding with a target-specific antibody, with MS analysis to identify proteins that interact directly with the purified antibody as well as proteins that may form a complex with the target protein.
“We suggest that at least one of these pillars should be used as a minimum criterion for claiming that a particular antibody has been adequately validated for a specific application,” the authors of the Nature Methods article wrote. “The use of multiple strategies would further strengthen this conclusion.”
The article also included recommendations for producers and users to ensure antibody reproducibility over time. For example, besides recommending that users of antibodies carry out at least one of the validation strategies described here in their own particular application or sample context, the article’s authors also suggested that users adhere to appropriate reporting guidelines and, at a minimum, include catalog number, lot number, and perhaps RRIDs (Research Resource Identifiers) to ensure that any antibodies used in their research can be identified unambiguously.
Similar advice—to adopt at least one of the five pillars, and then go further—was offered to producers, which were encouraged to provide as much additional information regarding the antibody as possible, such as antibody concentration in characterization assays, concentration of cellular extract or of the immunogen, details of the immunogen used for antibody production, antibody–epitope affinity, antibody isotype, buffer formulation, and the material data sheet. Among the producers that may be interested in these suggestions is Thermo Fisher Scientific, which extended financial support to the IWGAV in 2015 to encourage the development of standards and help overcome the challenges associated with antibody specificity and reproducibility.
“This publication is an important first step toward the development of widely accepted standards for validating antibodies and ensuring high quality and consistent antibodies for biomedical research,” said Dr. Uhlén. “We look forward to receiving feedback from the broader community of antibody users, publishers, funding agencies, and producers to help strengthen this initial proposal and ensure the reliability of these essential tools of biomedical research.”
“We recognize that input from all stakeholders, including funders, publishers, antibody providers, and the research community, will facilitate widespread adoption of our recommendations,” the authors of the Nature Methods article concluded. “Specifically, this wider community can provide critical insight into the timing for adoption of new proposals, the mechanisms used to enforce recommendations, and the specific responsibilities of each stakeholder as recommendations are implemented.”