: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
KP: As mentioned by the NIH, Nature and Science, the cornerstones of science advancement are rigor in designing and performing scientific research and the ability to reproduce biomedical research, as well as transparency and independent verification. At Bio-Techne, we want to do our part as partners with the research community to raise industry-wide validation standards for the benefit of scientists, advancing biological discovery, and to boost research reproducibility.
Antibodies are critical tools for life science research, and the acknowledgement that they are contributors to irreproducibility has now led to concerted efforts to tackle this issue. As one of the original innovators in protein and antibody-based research tools, reproducibility of research studies is something we at Bio-Techne have cared greatly about for the last 30 years. We believe using high-quality antibodies from a trusted source is key for ensuring reproducibility. This starts with expert antigen design, application screening and validation, production and quality-control processes. A highly experienced antibody development team is critical to all these steps. With over 50 years of combined antibody development expertise at our Bio-Techne antibody brands of R&D Systems and Novus Biologicals, we build and validate our antibodies and other key reagents with reproducibility top of mind. We make sure our antibodies give consistent results whether you carry out your experiments today or in 30 years’ time. We offer recombinant antibodies and recombinant conversion of hybridoma-derived monoclonals for those who want the ultimate in reproducibility and long-term supply security. In short, reproducibility is a key issue that we have taken very seriously at Bio-Techne for decades and not added on recently.
: 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?
KP: We routinely apply rigorous testing and validation of research antibodies we manufacture in-house or supply, using stringent validation workflows. When we bring in a hybridoma from an external source, we apply the same criteria as to our own cell lines. Validation strategies for antibodies can be complex, and we develop appropriate strategies for each target, sample and desired application(s) of interest. Our highly qualified antibody development and validation team is composed of experienced scientists who focus on a specific technique such as immunofluorescence, IHC, flow cytometry, Western blot, ELISA or functional assays. All data for each antibody is reviewed by a panel of scientists in a central review process to ensure only the highest performing clones are commercialized.
One important thing to note is that our antibody development and validation teams are distinct from our production teams. Any antibody transferred from development to the production team needs to meet the exact same validation and performance characteristics as specified by the antibody validation team during development. In short, if any clones or methods do not meet our rigorous specifications, we do not release them. This way, we are building reproducibility and robustness of methods into each quality clone.
At Bio-Techne, our aim is to provide only the highest quality affinity binders with the highest specificity, sensitivity and consistency. We have industry-leading validation standards, and access to the latest tools and technologies. For example, we are applying specificity testing methods using CRISPR knockout. We are probably one of the few companies that tests both the raw material lot as well as lots after the bottling process, even though this is resource intensive. We follow internationally accredited quality-management systems such as ISO 9001 to ensure our production and QC/QA processes are working well, and this ultimately helps with reproducibility.
: Do you have any advice, recommendations and/or best practices that scientists should follow when selecting and validating an antibody?
KP: When selecting an antibody, one piece of advice is to understand the provenance of the antibody you use. There are estimated to be two million research antibodies out there, from several hundred sources. It is often not feasible to buy all antibodies for a target of interest and test them side-by-side. One common approach researchers take when selecting antibodies is to follow the literature. However, these reagents may simply be the first available, and may not actually be the best reagent to use. Many studies use improperly validated, low-quality reagents, or antibodies that are later shown to be nonspecific. Our advice would be to select antibodies from a reputable vendor that has been demonstrated to be fit-for-purpose in the application and sample type of interest. We provide over 17,000 sample size antibodies to enable economical testing.
After selecting an antibody, we recommend performing proper context-specific validation of the antibody for the research study in question. Following the vendor’s guidelines in antibody usage is a good start; it would surprise you how many researchers don’t follow the instructions provided. We would also recommend use of caution when employing an antibody in an application that is not recommended by the supplier. An antibody that has been shown by a vendor to work beautifully in Western blot, but has not been shown to work in other techniques, would require validation on the customer side for that new application. We recommend the use of proper positive and negative controls. There are different ways to validate an antibody depending on the research question and target type (e.g. transcription factor, cytokine or a PTM). Therefore, there isn’t a one-size-fits-all approach to antibody validation. Don’t forget that vendors like Bio-Techne provide expert technical support and you can have contact with the team or scientist who produced an antibody.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
KP: At Bio-Techne, we are fully behind current efforts to raise antibody validation standards in the industry, and collectively tackle the reproducibility problem. One of the key programs we are engaged with are the antibody validation workshops organized by the Global Biological Standards Institute (GBSI). The five conceptual validation "pillars" published in Nature Methods by the International Working Group on Antibody Validation (IWGAV) provide a helpful framework for discussion of application-specific standards for validating antibody specificity. These include genetic strategies, independent antibody strategies, orthogonal strategies, expression of tagged proteins and immunocapture followed by mass spec (IP-MS). We agree with IWGAV recommendations, including the need for application-specific and context-specific validation of antibodies. We are working with the stakeholders on developing these guidelines further to ensure recommendations are practical, workable and usable.
Some of the guidelines may be unrealistic for smaller vendors, due to technological or economic limitations. Let’s take the genetic validation pillar; to make a CRISPR-based knockout of all genes in several standard cell lines would be a huge undertaking for any single manufacturer. Right now, there are only a few leading vendors such as ourselves, who have committed to applying CRISPR knockout studies for specificity validation, but the issue of cost, speed and scalability remains. It should also be borne in mind that there are technical challenges with gene-editing technology, appropriate cell lines, and its inapplicability for certain targets such as epigenetic targets or PTMs.
Besides development of validation method standards by producers, we believe that there is a huge role to play by all the different stakeholders: from journals, funding agencies, antibody databases, research tool identification initiatives (e.g. RRIDs) and institutions, to help promote antibody training for researchers or more detailed materials and methods such as providing all clone and vendor information or full-size Western blot results.
: What else is your company doing to address reproducibility?
KP: Besides our own efforts, Bio-Techne has been a key supporter of many initiatives related to reproducibility from different international groups. We are sponsors of the Reproducibility Project, which set out to replicate key experimental results from 50 high-impact cancer biology studies using experts from the Science Exchange network, and reported results recently. F1000Research is a peer-reviewed Open Science publishing platform where we cover publication fees for any article submitted to the Antibody Validation channel. Another important initiative we support is from Antibodypedia in Sweden, where researchers use genetic or other strategies proposed by the IWGAV to prove antibody specificity in a particular application(s). The results are submitted to the initiative and made publicly available on Antibodypedia. We have also started working with other partners such as Biocompare, CiteAb, The Antibody Registry, as well as the European Monoclonal Antibody Network, and are excited to be involved with a new series of researcher antibody validation training workshops that are taking place in different countries. We are also offering the know-how of our expert scientists in seminars and through provision of online resources.
Author Bio: Karen started Novus Biologicals in the summer of 1996 to be a company that licenses, produces and markets antibodies for niche and emerging areas of research. She successfully ran Novus Biologicals with consistent growth for 18 years out of Littleton, CO. She sold Novus to Bio-Techne in July 2014 to become one of the brands that greatly expanded the antibody portfolio for customers working in tandem with R&D Systems brand. Karen continues her commitment to her work as the Vice President of the Antibody Business Unit and Digital Marketing with Bio-Techne.