: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
CA: High-quality antibodies directly contribute to high reproducibility rates within life science research and scientific advances. These critical reagents must be properly used by researchers to obtain the desired quality and consistency of generated data. These two points are essential for the future, as they impact basic research, pre-clinical therapeutics, in vitro diagnostics, food safety, and biosecurity.
To accomplish this goal we must foster an environment where continuous improvement of both antibody quality and validation standards is commonplace. We must collectively do more to improve the manufacturing process and adopt robust validation standards as they are developed. But the global resolution of problems pertaining to antibodies will require fundamental changes in how many stakeholders approach the topic, including manufacturers, resellers, researchers, funding agencies, journals and universities where most training of scientists occurs.
: 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?
CA: Every lot of every antibody is tested in a consistent manner before it is sold. We balance the rigor of antibody validation with the value the antibody has to the researchers. For instance, a newly developed antibody may be initially validated by Western blotting using recombinant proteins or lysates that overexpress the target protein. But as the antibody is used more by researchers we increase the rigor of validation to include native cells or tissues that may have been treated by genetic manipulation or drug studies or presented as multiple lysate panels with expression levels known based on proteomic studies of the target protein. This information is then correlated with the observed band intensity after antibody staining as part of our validation strategy.
Antibody validation is both lot specific and immunoassay specific, meaning each newly manufactured lot or batch or antibody is validated independently, and that the validation strategies developed, say, for Western blotting may not necessarilyy apply to immunohistochemistry or ELISA. Furthermore, we perform every validation experiment with the appropriate negative and positive controls and we strive to ensure that this information, in its entirety, is communicated to the researcher to foster transparency so that results can be repeated when protocols are precisely followed by users.
: Do you have any advice, recommendations and/or best practices, scientists should follow when selecting and validating an antibody?
CA: Today, more than ever, researchers must be able to discriminate ‘high quality’ antibody manufacturers from other providers that take risky validation shortcuts, or worse, do not perform validation of any type, especially in a global marketplace. Researchers should look for manufacturers that transparently convey antigen, testing and release data to the researcher, including in which assays the antibody is demonstrated to show acceptable performance. Manufacturers should report either on their website or on a certificate of analysis, the nature of the immunogen, screening criteria during antibody development, and lot-specific validation data. Researchers should consider any vendor not presenting this information as a red flag or warning concerning the potential performance of an antibody. Researchers must carefully review this information and, when necessary, independently validate an antibody before commencing with experimental research.
Manufacturers of high-quality antibodies participate in scientific meetings and interact with research scientists at every opportunity. This contact allows all manufacturers to adapt and change as the needs of researchers for novel antibodies and validation methods likewise change. Other companies that do not keep current with validation methods or data transparency may be providing astute users an insight into their approach to manufacturing, reselling and quality.
Always look for the inclusion of the appropriate positive and negative controls in validation experiments and discount the value of truncated data, for instance, the digital cropping of a Western blot image to only the molecular weight range of the target protein. When data of this type is presented the researcher has no way of knowing the degree of off target binding for the antibody, which may be why the image was cropped.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
CA: The meeting organized by the Global Biological Standards Institute (GBSI) in September of 2016 and the preceding meeting of the International Working Group on Antibody Validation where the “pillars of validation” were proposed were both steps in the right direction. These meetings brought together a diverse group of stakeholders: academic researchers, industry, funding agencies, teaching universities and journals. Global change will take a community effort by all of these stakeholders. If these efforts remain focused on a broad-based approach to improvement, then they will likely succeed, be implemented and result in significant improvement regarding data reproducibility. If, however, the focus of change becomes limited to one or a few stakeholders, or if the recommendations become skewed to favor particular users or commercial interests or manufacturers, then the outcome will likely not be attained.
: What else is your company doing to address reproducibility?
CA: Rockland Immunochemicals is an active participant in several efforts designed to improve antibody validation, including efforts to set assay-specific validation standards, and establish certification and training programs. As a leading manufacturer of antibodies, we recognize how important antibodies are as critical reagents used by life science researchers.
But antibodies are more than just research tools. Antibodies are transformative agents capable of curing diseases and saving lives, ensuring the safety of the food chain, and protecting society from biological threats. Our overarching goal of continuous process and quality improvement is designed to ensure that all forms of antibodies, polyclonal, monoclonal and recombinant, result in the expansion of antibody-based technologies. We recognize that novel fit-for-purpose antibodies are required with highly precise properties for use in novel platform technologies, to identify new biologically significant targets, to study orphan diseases, and to explore rarely studied species. We can meet and exceed these goals if we work together. We are committed to helping shape this bright future.
Author Bio: Carl Ascoli, Ph.D. is the Chief Science Officer of Rockland Immunochemicals and has more than 30 years of academic and industrial experience and a broad knowledge in virology, immunology, host-agent interaction, autoimmune diseases, cancer biology, antibody production and engineering, protein purification and DNA recombinant technology. Dr. Ascoli joined Rockland Immunochemicals in 1991 and began to apply his skills in molecular immunology to solve problems for academic collaborators who required the production of highly specific polyclonal and monoclonal antibodies for research. For more than 20 years Dr. Ascoli has established collaborations with principal investigators at academic and research throughout North America, Europe and Japan.