: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
DGW: Antibodies are some of the most critical reagents used in life science and medical research. However, the use of poorly characterized antibodies can generate significant problems–compromising research projects and costing valuable time and money. According to an article published in Nature in 2015, an estimated $350 million in the United States and $800 million worldwide is wasted each year as a result of nonspecific or poorly validated antibodies contributing to failed or unreproducible experiments. While wasting precious research budget is never acceptable, it is becoming increasingly important during times of funding uncertainty that vendors and scientists align to address this issue.
While the issue of antibody validation standards has been discussed for quite some time, 2016 represented a major shift in the research community’s approach to addressing the challenge. In September, Nature Methods published the first proposed strategies to create standards for validating antibody specificity, brought forth by the International Working Group for Antibody Validation (IWGAV). Later that month, the Global Biological Standards Institute (GBSI) held the Asilomar Antibody Validation Workshop, where key stakeholders across the industry discussed strategies for validating antibodies, building on the priorities outlined in the Nature Methods piece.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
DGW: The proposal and community discussion are the start of a long process that will require continuous cultivation and collaboration. Thermo Fisher Scientific has already adopted the validation standards for our antibody portfolio in line with the recommendations of the International Working Group for Antibody Validation (IWGAV). Using these new Thermo Fisher Scientific specificity validation standards, Invitrogen antibodies are being tested for both target specificity and robust application validation aligned to a variety of applications such as imaging, flow cytometry and Western blotting.
To ensure that an antibody is binding to the correct target, we are testing our antibodies using one or more of the following four Thermo Fisher Scientific specificity pillars:
- Genetic modification where antibodies are tested using cell lines following knockdown or knockout technologies
- Independent antibody verification in which measurement of target expression is performed using two differentially raised antibodies recognizing the same protein target
- Functional or orthogonal testing in which functional activity or changes of target levels are measured in different cells or following cell treatments
- IP-mass spectrometry, where antibody targets are identified by mass spec following isolation by immunoprecipitation
The specificity validation data will be combined with application validation in a relevant biological model system. As we expand specificity and application testing of our extensive portfolio of antibodies, data showing specificity verification will be increasingly seen on our product detail pages and information sheets.
: Do you have any advice, recommendations, and/or best practices, scientists should follow when selecting and validating an antibody?
DGW: When scientists are selecting antibodies to use in their particular experiment, a number of considerations must be taken into account:
- Try to find an antibody that fits the applications and species reactivity required. Often it is tempting to try to pick an antibody that does everything, but sometimes this does not provide an antibody that works the best in the most important applications.
- Over what period of time is the antibody going to be needed? If the antibody is going to be needed over many years with multiple purchases, then you may want to consider finding a monoclonal. While polyclonals can be excellent reagents, there is a slightly higher chance the antibody properties can change over time.
- How has the antibody been validated? Does the supplier show robust validation data verifying the applications for which they claim the antibody works?
- What experimental models have been used to validate the antibody? Have the appropriate cell line(s) been used? Are the proper experimental controls used?
- Has the antibody been used in publication? How many other people have used the antibody successfully to answer relevant biological questions?
- Has the antibody been confirmed to be specific? Has the supplier shown appropriate controls to confirm the antibody is detecting the correct protein of interest? Even better, has the antibody been shown to be specific by one of the IWGAV specificity pillars?
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
DGW: The current efforts underway for the antibody reproducibility problem are a major step forward in solving the antibody reproducibility crisis, but it will take the entire community to embrace these new standards for proper implementation. Researchers will need to carefully consider which antibody companies are providing enhanced validation data to ensure antibodies are binding to the right target and are validated for proper application use. Research journals and funding organizations will need to require researchers to provide assurance that the antibodies used in their research and research proposals have gone through this additional rigor in validation. Antibody companies will need to expand their validation testing on the antibodies they provide and seamlessly provide researchers with the data and information required by journals and funding agencies to show the antibodies have been thoroughly tested for specificity and application validation. By working together, these groups will be able to ensure the antibodies used in their experiments are of the highest quality and can be comparable between research studies.
: What else is your company doing to address reproducibility?
DGW: Thermo Fisher Scientific is committed to working closely with the research community to provide them the testing criteria and data to give researchers the confidence that the antibodies provided have been validated for both specificity and application validation. We have begun to work through our extensive Invitrogen antibody portfolio to test our antibodies in one or more of the validation pillars, in addition to the extensive applications validation and rigor already done across our portfolio. We will continue to work to adopt antibody validation standards proposed by the research community to ensure researchers are spending their hard-earned research dollars on antibodies that work for their intended research application, thereby returning millions of dollars back into the research community.
Author Bio: Dara Grantham Wright serves as the Vice President and General Manager for the recently formed Antibodies and Immunoassays Business Unit in the Biosciences Division. The business unit is composed of the legacy eBioscience Business Unit (Affymetrix) and the Immunoassay Systems portion of the Protein and Cell Analysis Business Unit.
Prior to its acquisition by Thermo Fisher Scientific in early 2016, Dara led the Affymetrix eBioscience Business Unit, which is composed of a large portfolio of reagents and kits used for cell analysis.
Dara has nearly 20 years of global experience developing and commercializing products for life science and clinical diagnostics applications. Ms. Wright holds an MBA from San Diego State University and a double B.A. in Biology and Spanish from Willamette University.