Q&A Part 5: Industry Leaders Discuss Their Efforts to Address Reproducibility

This company was founded by scientists, and we have addressed reproducibility since the company’s inception in 2001 so we don’t need to address reproducibility in the same manner perhaps other antibody companies must. When we started the company we knew reproducibility was of utmost importance. We created a thorough scientific database that documents every step we’ve made in the production and validation processes. This thorough and highly organized data allows us to reproduce the antibody, effectively resulting in lot to lot consistency and reproducible data. All this in-house reproducibility allows for expert technical support and ease of troubleshooting for scientists. We can share original data and the protocol used specifically for the target in mind. It truly benefits the scientist and science to purchase directly from a manufacturer. You know exactly what you’re getting when you do.

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.

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.

Atlas Antibodies has always had high requirements on antibody validation and reproducibility. Right now, we are improving the way we communicate all validation data and the stringent quality-control procedures we follow to secure reproducibility. We have started to implement the formal validation strategies suggested by the IWGAV, in an application-specific manner. This formal validation will be illustrated by icons representing the different validations methods: genetic, orthogonal, or independent antibody strategies or expression of tagged protein strategy. The validation icons will serve as visual help for scientist to identify antibodies that comply with the formal validation strategies.

Atlas Antibodies strives to continuously improve our service to our customers and to help scientists choose the best antibodies for their research. As always, transparency remains of great importance to us.

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.

The most important thing CST can do is to continue to work hard to maintain our industry leadership and efforts in antibody validation and overall quality. Our customers know our quality reputation, and we will maintain that. We are constantly improving our methods and technologies to do things better. Whether it’s the use of CRISPR technology or mass spectrometry-based methods, we continue to expand our capabilities to deliver to our customers the best products we possibly can. We will continue to help educate our customers by making available protocols, tutorials and scientific material on our website and by different outreach campaigns to help scientists succeed in their projects.

Actively participating in the debate and contributing to solve the reproducibility problem as part of the scientific community is very important. We will continue to do so. But we also hope to help create an industry forum or consortium where the reproducibility problems and solutions can be debated to help elevate the quality of our industry as a whole. As these changes take hold, the research community that we are a part of will benefit and we’ll begin to see new scientific discoveries made more reliably and in less time.

We are looking to become more directly involved in the organized movements that are addressing antibody standards, including the evaluation of test methods that were recommended in these forums. Also near term, through our ongoing practice of customer interviews and feedback we will improve product search and usage experiences. And longer term, we certainly intend to pursue ways to address this issue more upstream, improving the criteria we use in new product selection and innovation.

Miltenyi Biotec has shifted all the processes for generating antibodies for flow cytometry toward making them recombinantly. They are sold under the brand name of REAfinityTM Recombinant Antibodies. Recombinantly generated antibodies possess highly defined characteristics and are produced under standardized culture conditions leading to products that are reproducible in nature. This saves users the time associated with standardizing analysis and decreases the financial burden associated with working with inconsistent reagents. Recombinant technology also opens up myriads of possibilities to improve antibody properties. REAfinity Antibodies provided by Miltenyi Biotec are engineered to lack background binding and share a single isotype. This solves the two common problems associated with flow cytometry: nonspecific signals and complicated experiment planning with multiple antibody isotypes.

Several of my colleagues at Bio-Rad and myself are actively involved in the efforts of the Global Biological Standards Institute (GBSI) to solve the reproducibility crisis by trying to define antibody validation standards in qualitative and quantitative terms. We participate in regular meetings, discussions, and working groups led by the GBSI. We are also looking for ways in which we can improve our own antibody validation, for example by using gene disruption technologies like CRISPR to eliminate expression of the antibody target. Finally, we are working with our customers so that they are aware of the importance of using high-quality reagents, such as validated antibodies, in properly controlled experiments.

Externally, BioLegend supports a variety of reproducibility and transparency programs, both financially and by contributing products. For example, we have been early supporters of the Resource Identification Initiative, led by Anita Bandrowski, where all of our products are matched to an RRID # (Research Resource Identifier). We’ve also been supporters of the GBSI and are working with them specifically on setting standards for flow cytometry antibodies.

Internally, we have stringent quality systems that monitor each production lot and strive for continuous improvement over our already high standards. During validation, we always look to introduce new technology, new assays and to develop more relevant model systems and controls. The data generated is made publicly available through our website. We have also begun citing contra-indicated applications when we have demonstrated an antibody is not suitable for a particular application. We track and list publications that use our products on our website. Reviews of our products are encouraged and visible through our website. Product stability is continually reviewed via real-time studies in addition to any accelerated stability studies. Our shipping and distribution networks are continually examined to find ways to minimize the chance of delays in-transit to mitigate any associated product issues. Our Technical Service Scientists work to track down the root cause of every potentially problematic inquiry to identify any quality issues that need to be addressed.

BioLegend’s antibody offering is overwhelmingly monoclonal and manufactured in-house. Focusing on mAbs allows us to ensure a long-term reproducible supply. We are also assured that our internally manufactured material will meet criteria to seamlessly pass into our downstream processes that transform antibodies into a variety of different formats and fluorescent conjugates. These modified versions of our mAbs undergo further QC to ensure they function as expected in multicolor panels and multiplex assays.

In addition to the strategies described previously, GeneTex is also taking a longer view to addressing the antibody reproducibility challenge. Consistent with the comments of Bradbury and Plückthun,¹ GeneTex is currently evaluating different platforms for the production of recombinant antibodies. We are convinced that this is the direction that the commercial research antibody market is heading, and GeneTex views this as a goal to be pursued over the next several years.

Until that time, GeneTex will continue to expand our knockout/knockdown program. For our antibody reagents that are directed against post-translational modifications or against proteins that are not expressed in easily transfectable cell lines, we will continue to design individual plans for specificity assessment incorporating drug treatments, point mutants, peptide blocking and known positive and negative control cell lines. GeneTex will also further refine immunogen sequence selection so that our antibodies will be specific for particular isoforms or proteins from families with high sequence identity.

The industry is at a point where our customers are demanding better validation of the antibody reagents that are offered. Those companies that fail to meet these higher expectations will lose the trust of the research community and will not survive. At GeneTex, we accept this challenge and will pursue the strategies described here to continue to earn the business and loyalty of scientists who depend on reliable antibodies to move life sciences discovery forward.

References

1. Bradbury, A. & Plückthun, A. Nature 518, 27-29 (2015).

LI-COR is leading the way to improve reproducibility for researchers that do Western blotting, an application that, of course, requires both validated primary and secondary antibodies. Following JBC’s lead in releasing new requirements to help researchers generate and submit more reproducible Western blot data, we have developed new methods and protocols to assist LI-COR Odyssey® users in successfully addressing these new requirements. We have found in our own studies that these new methods can help researchers generate more reliable, reproducible Western blot data.

New protocols for generating more reproducible, reliable Western blot data explain how to:

• Validate a housekeeping protein as an internal loading control for Western blot normalization.
• Use a housekeeping protein, pan/phospho strategy, or REVERT™ Total Protein Stain as an internal loading control for Western blot normalization.
• Find the combined linear range for accurate detection of both the target protein and internal loading control.

As mentioned previously, the reproducibility crisis is in part derived by different methods and reagents from various sources. Sino Biological strives to make sure our antibody reagents meet the highest criteria in the field. A Certificate of Analysis is issued for each lot of product, which includes:

• Product information: Product name, catalog number, lot number, immunogen, clone ID, Ig type, formulation, preparation process;
• Antibody usage guide: Specificity, application, storage;
• QC-approved images;
• Lot-specific test results that are helpful for using the product

We guarantee our products for approved applications. Furthermore, our technical support department is available to address researchers’ special requests. We have put considerable effort into tackling the reproducibility problem, not just for our products but also for reagents from different sources.