Antibodies are some of the most critical research reagents used in the lab. Poor specificity or application performance can significantly frustrate the ability to obtain good results, which can cause critical delays. Underperforming antibodies result in a lack of reproducibility, wasting time and money. In other words, researchers need antibodies that bind to the right target and work in their applications every time. To help ensure superior antibody results, we've expanded our specificity testing methodology using a two-part approach for advanced verification.
Lack of access to appropriate antibodies to detect post-translational modifications is a recognized problem for researchers. Learn more about the reagents with improved specificity.
In collaboration with Nature, learn more about Thermo Fisher Scientific’s commitment to antibody validation and performance. This series of three articles explores the reproducibility crisis, protocols and standards, and our specificity testing methodology with data from numerous antibodies and pathways.
This helps ensure the antibody will bind to the correct target. Our antibodies are being tested using at least one of the following methods to ensure proper functionality in researcher’s experiments. Click on each testing method below for detailed testing strategies, workflow examples, and data figure legends.
These tests help ensure the antibody works in a particular application(s) of interest, which may include
(but are not limited to):
Most antibodies were developed with specific applications in mind. Testing that an antibody generates acceptable results in a specific application is the second part of confirming antibody performance.
Thermo Fisher Scientific is committed to adopting higher validation standards for the Invitrogen antibody portfolio. We have implemented additional specificity tests to help ensure the highest confidence levels in our products. You can identify the products that have already undergone this testing with the Advanced Verification badge, shown above. This badge can be found in antibody search results and at the top of product webpages. The data supporting the Advanced Verification status can be found in the product specific data galleries. To learn more about our testing standards, please visit Invitrogen Antibody Validation.
As seen in Science, use these tips for setting up, designing, doing, and troubleshooting your experiments.
Learn more about our two-part validation testing approach in this easy-to-read, one-page document. It summarizes our comprehensive verification testing process and describes how we provide a new standard of confidence for our customers.
In collaboration with Wiley publishing, learn more about what suppliers, publishers, and others in the scientific community are doing to address antibody reproducibility problems.
Read the app note to learn how Thermo Fisher is validating antibodies in the EGFR pathway, using CRISPR knockout and other specificity tests to verify antibody performance.
Learn which criteria to consider when choosing a histone PTM-specific and how we’re validating these antibodies with the most appropriate tests, including peptide array, ChIP and ELISA.
Reproducibility and antibody validation standards are two significant challenges facing scientific researchers today. The panel will discuss current practices to find a consensus for verifying antibodies used in common laboratory techniques.
Every year, millions of dollars are wasted on poorly characterized and performing antibodies. During this panel forum, key researchers in the antibody community came together to address this reproducibility crisis. They discussed the development of validation* testing standards to ensure proper characterization and consistency for antibodies in the laboratory (this panel originally took place on May 30, 2017).
Aled Edwards, CEO of Structural Genomics Consortium and on the faculty of the University of Toronto in Ontario, Canada. He’s also a member of the International Working Group for Antibody Validation, IWGAV.
Anita Bandrowski, Scientific Lead at the New Science Information Framework at the Center for Research and Biological Systems at the University of California at San Diego. She’s also the Founder and CEO of SciCrunch and she is also an IWGAV member.
Paul Wallace, Professor of Oncology and the Director of the Flow and Image Cytometry Facility in Roswell Park Cancer Center and Institute in Buffalo, New York. He’s also an Associate Professor of Pathology at the State University of New York in Buffalo.
Matt Baker, Director of Strategy and Partnering for Antibodies and Immunoassays with Thermo Fisher Scientific.
John Rogers, Senior R&D Manager for Mass Spectrometry Reagents in the Protein and Cell Analysis Business with Thermo Fisher Analytics.
Christoph Hergersberg (Moderator), Vice President for R&D for Protein and Cell Analysis and for Antibodies and Immunoassays with Thermo Fisher Scientific.
Edwards: Antibody reagents have been of questionable characterization, for the past 30 or 40 years. It’s a very difficult problem to solve and involves sorting through the complexities of market forces and scientific incentives. It really came to the forefront 5 to 6 years ago when pharmaceutical companies started to publish the results of their internal experiments in academic literature and they found that a large number of early discovery projects couldn’t be reproduced.
Bandrowski: Another thing that adds to this whole reproducibility dilemma is the way researchers are publishing their results. They’re getting these great results, but the way they publish, it doesn’t allow other people to actually reproduce those results. This is one of the simplest things that we’re not doing as a scientific research community. It was addressed a few years ago in the Vaseleski** paper, essentially showing that over half of the antibodies that are published can’t be tracked down to the actual antibody that was used.
Edwards: Recently some new, game-changing technologies emerged that have made it possible to put quantitative metrics on the quality of an antibody, such as CRISPRs to create knockout cells and mass spectrometry. This made it possible to create standard community criteria for antibodies that the scientific community can adopt.
**Vasilevsky NA, Brush MH, Paddock H, et al. On the reproducibility of science: unique identification of research resources in the biomedical literature. Abdullah J, ed. PeerJ. 2013;1:e148. doi:10.7717/peerj.148.
Baker: As a major supplier and manufacturer of antibodies, reproducibility is a key issue to Thermo Fisher Scientific, as we work with researchers in the science community. Customers want confidence that their antibody will work, so we’re using new technologies and focusing to confirm specificity.
We’ve adopted a two-part systematic approach to test our portfolio, validating for both functional application and advanced specificity verification. Specificity testing involves 9 test methods; Immunoprecipitation/mass spectrometry, Knockout, Knockdown, Independent antibody verification, Cell treatment, Relative expression, Neutralization, Peptide array, and Orthogonal method. In conjunction with each approach, we’re including testing data for each antibody for transparency and reproducibility.
Edwards: With mass spec applications, it’s a very high statistically significant result when you get the peptide from your protein, or a series of peptides from your protein, in your immunoprecipitate. You can’t fake that. So there are methods that are absolute. When you make a CRISPR knockout, if the genetics are right, it is absolute that protein’s not there.
Edwards: Simply reproducing experiments is not the aim. The aim is to make sure that the antibody has quantitative and standard operating procedure kind of characterization. CRISPR knockout is the best along with mass spectrometry, both with quantitative results. I think all of your testing methods will be a tremendous “flag” that you plant in the community as to what is a proper validation of an antibody.
Bandrowski: Another thing I really like is releasing more data. If a researcher can be more comfortable by seeing a whole pile of data, there’s a different value that can be put on that.
So if you can see the comparison between here is what the company did, here is what I’m doing and there is something wrong, so being able to just see that, being able to have that full data I think is incredibly valuable resource for the researchers.
Wallace: If we’re talking about validated reagents with protocols using CRISPR cell lines or the other methodologies described, it would be nice to include specific protocols so that if I purchase this particular antibody or I was to use this particular reagent and I follow this procedure that I could actually get that result. There’s certainly a lot of variation from one laboratory to another, but a protocol that we could follow that worked with a particular reagent, would allow me to feel comfortable making modifications to my procedure to see if I could improve it.
Lastly, there are a lot of antibodies out there that will work really well in a Western blot that completely fail when it comes to immunohistochemistry. And likewise, there are antibodies that work well by flow by don’t work in immunohistochemistry or don’t work when you do a particular technique. When you discuss how you will validate, it isn’t just in one methodology or one technique. It really is a variety of techniques to prove specificity, similar to your two-step testing approach.
Wallace: I would start with the specific cell and my question, and then select the antibodies that I’m going to need to address that specific question. Next I search my options on manufacturers’ websites to see what flow clones are available as I put my panel together. If I saw that my clone had been validated, would work well in flow cytometry and included a protocol describing is how to use it, that would definitely attract me to that particular reagent.
Then I determine what the fluorochromes will be, and the cross talk between one reagent and another reagent depending on how highly it’s expressed. The first step is to titrate it and validate the panel. As I put that panel together, I’d run a number of controls like FMOs, or fluorescence minus ones. This would validate that the panel is working well, meaning I’m getting the best separation in all the populations. And having protocols and examples of where this particular antibody worked, would speed up the process.
Bandrowski: There aremany initiatives that will have to change which will take time to work their way through the community. In 2016 the grant criteria for review changed, and has been updated, moving towards rigor. Over the next five to ten years we’ll see these grants moving towards the authentication of biological reagents, specifically antibodies, cell lines, etc. This is a really important thing because it involves the whole community.
I would hope that some of the other funders would follow suit with the NIH, including journals which are also taking notice and moving towards reproducibility. By tackling the identification of reagents, we can align the papers with the grant review guidelines towards rigor. This will be a multi-year process to get the community on board, however there are things that every researcher can do today. Utilize research resource identifiers, for all their antibodies and share data when possible.
Thermo Fisher Scientific is committed to adopting validation standards for our Invitrogen antibody portfolio that are tested for both target specificity and functional application. By supporting the International Working Group for Antibody Validation (IWGAV), and adopting their recommendations, we’re doing our part to ensure reproducibility and proper functionality in the scientific community.