Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack

Millions of animals are used for the routine production of antibodies for research, monitoring, diagnostics, and therapeutics, although the true magnitude of use is unknown. The EU Directive on the protection of animals used for scientific purposes (2010/63/EU) requires that animals are not be used for scientific purposes where a non-animal alternative exists. Animal-friendly affinity reagents (AFAs) from non-immunised sources and produced by phage display are mature and available to replace animal immunisation methods for antibody production. In line with Directive 2010/63/EU, an EU-wide replacement programme must now take precedence. In this Opinion article, we outline recommended actions that must be prioritised. The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported. The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported. A young woman visiting her local supermarket to do her weekly shopping knows that, in this conscientious-consumer-driven society, she will be overwhelmed with choices that allow her to make informed decisions about her own environmental footprint, maintain a healthy lifestyle, and patronise non-exploitative industries. The information that helps her do so is clearly visible on the packaging of the shampoo especially formulated for sensitive skin and the nutrient-supplemented, low-sugar, fair-trade cereal that she chooses as she browses the aisles. However, on this occasion the main reason for her visit is to pick up a pregnancy test that will help her plan her future. She knows that the three items she has just selected will all contribute to protecting her family and the environment they live in, but does she know that, despite the reassuring information on the packaging, she has just picked up three items that still use animals? How is that possible? The shampoo conforms to EU cosmetics regulation 1223/2009 by not being tested on animals, the cereal is animal-product free, and the pregnancy test is a simple over-the-counter diagnostic test designed for home use. What the information on the packaging does not tell her about is the hidden use of animals in an affluent, US$80-billion industry that creates millions of animal-derived monoclonal and polyclonal antibodies (see Glossary) to detect a vast range of molecules indicative of state of health, safety, or the environment. Antibodies are used, for example, as an indispensable link in the health-care chain, contributing to enhanced patient care and reduced public health costs. Antibody-based tests are used to diagnose and monitor infectious or chronic diseases, to manage oncology treatment, or, in rapid-test format, often available over the counter, to monitor fertility, ovulation and pregnancy, sexually transmitted diseases, substances of abuse, and performance-enhancing drugs. Also, as a vital tool to safeguard the environment and consumer industry, antibodies may be used to detect or extract the alarming number of potential chemical contaminants, allergens, microbiologicals, natural toxins, food constituents, pharmaceutical and veterinary drugs, hormones, metals, agricultural contaminants, and pesticides that can exist in our food, beverages, and water. They can predict the safe limit for a potentially harmful ingredient in a cosmetic or household product or control the quality of biological agents such as vaccines or botulinum toxin. The tests that are employed in these examples appear to be far removed from animal experimentation since no animals were directly tested on. However, the molecule to be detected is repeatedly injected into the animal, initiating a hyperimmune response. Months later, antibodies are extracted and incorporated into an in vitro, animal-free test for the detection of the molecule. So in reality we are not replacing animals but simply substituting methods in which the animal use is direct for other methods where the animal use is buried several layers deep in the production process, and our ultimate aim, to replace needless animal use, is not achieved. Also overlooked are the sheer numbers of animals that are sacrificed to produce antibodies and the associated and unaccounted-for animal-welfare issues [1Reardon S. US government issues historic $3.5-million fine over animal welfare.Nat. News. 2016; (Published online May 20, 2016)https://doi.org/10.1038/nature.2016.19958Crossref Google Scholar, 2Leenaars P.P. et al.Assessment of side effects induced by injection of different adjuvant–antigen combinations in rabbits and mice.Lab. Anim. 1998; 32: 387-406Crossref PubMed Scopus (70) Google Scholar]. Measures to improve animal-welfare standards by reducing or refining animal-derived antibody production methods were initiated in 1998 following the publication of a statement by the European Centre for the Validation of Alternative Methods (ECVAM) scientific advisory committee. This statement recommended that, following the immunisation protocol, the propagation of monoclonal antibodies in the ascites of the abdominal cavity was no longer scientifically necessary. Accordingly, competent authorities in European Member States responsible for the implementation of EU legislation regarding the welfare of animals used for scientific purposes should now no longer authorise project license applications proposing antibody propagation by the ascites method. Recommendations for refining methods for producing polyclonal antibodies were also published. Remarkably, workshop reports that were distributed to all EU Member State competent authorities highlighted advances in novel recombinant DNA-based technologies and direct cloning into plasmids. These reports envisaged that, ‘in the near future’, phage display, an advanced technology capable of producing binders ‘without prior immunisation of B cell donors (would) avoid the need to use living animals’ [3Marx U. et al.Monoclonal antibody production.ATLA. 1997; 25: 121-137Google Scholar, 4Leenaars M. et al.The production of polyclonal antibodies in laboratory animals.ATLA. 1999; 27: 79-102Google Scholar, 5European Centre for the Validation of Alternative MethodsStatement on the Scientific Acceptability and Practical Availability of in vitro Methods for the Production of Monoclonal Antibodies.1998Google Scholar]. So looking back, this discrepancy with reality is not easy to understand. Eighteen years later, immunised animals are still heavily relied on for hybridoma (monoclonal–with subsequent amplification in vitro), polyclonal, and even some recombinant antibody production methods. There are ∼123 EU companies offering ∼714 000 catalogue antibodies produced either in-house or through international collaborations (more than 2.5 billion worldwide). Furthermore, undetermined numbers of antibodies are generated through custom-made production by companies and research institutes. Demand is set to increase because the number of antibodies that could be generated in the future, including variants, in all different immunoassays, appears to be limitless. The human genome has 20 000–25 000 protein-encoding genes. Due to alternative mRNA splicing and post-translational modifications, the number of proteins and the potential for the generation of antibodies exceed this number tenfold. It is not uncommon to see hundreds of antibodies generated for the same target (e.g., more than 900 for P53 [6Taussig M.J. et al.ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.Nat. Methods. 2007; 4: 13-17Crossref PubMed Scopus (210) Google Scholar, 7Hust M. et al.A human ScFv Ab generation pipeline for proteome research.J. Biotechnol. 2011; 152: 159-170Crossref PubMed Scopus (102) Google Scholar]). Larger numbers of animals are required to increase the chances of success if the desired antibody is expected to distinguish closely related isoforms within or across species or other subtle changes (e.g., phosphorylated versus non-phosphorylated targets). Yet the actual number of animals used to generate these antibodies is not known. According to the latest EU statistics on the number of animals used for scientific purposes [8European CommissionSeventh Report from the Commission to the Council and the European Parliament on the Statistics on the Number of Animals Used for Experimental and Other Scientific Purposes in the Member States of the European Union COM(2013)859/final.2013Google Scholar], animal procedures (see Glossary) for antibody production are immersed into 3 different categories, that includes all animal procedures for ‘production and quality control of products and devices for human/veterinary medicine and dentistry’ (categories 2.4 and 2.5) and ‘other’ (category 2.9). While the combined total number of animals used in these three categories is 2.7 million, or 23.18% of total animal use, it is not possible to determine what proportion of this animal use is attributed to the production of antibodies alone. Only two of the EU Member State countries publish this information in their own national statistics. In 2013, the UK reported the use of 9522 animals to produce antibodies (1433 monoclonal and Statistics of Scientific on of the combined total use of animals in categories and the number used in cosmetics a on animal use was In the same The used more than the number of animals to produce with animals and over or of the combined total use of animals in these three These statistics no into the on animals because antibodies are also in countries where animal welfare is and into the Directive 2010/63/EU on the protection of animals used for scientific and in Parliament and Council of the European 2010/63/EU of the European Parliament and of the Council of on the of Animals Used for Scientific Scholar]. The are the the use of animals in scientific research and are in national and international legislation the use of animals in scientific The Directive to and the of non-animal alternative that could the same or of information as from animal the of replacement and of the Directive that a scientifically or testing not the use of live animals, be used of a and that an animal should not be out if or testing for the not the use of a live animal, is the legislation of the It requires that competent in Member and authorise including using methods for diagnostics, or production, such as the commercial production of The Directive also that Commission should also the reduction and of the use of animals in as of the to alternative to the use of animals, the of the are in of the Directive and and the and use of alternatives to procedures including in the of and research and the of alternative Union as a for the of information on the of alternative and public and information on alternative and their state of and and all in industry, consumer and animal-welfare with a to the international and of alternative has over the 20 years and antibody production by animal-friendly methods has to a of scientific that animal immunisation numbers of different animal affinity reagents (AFAs) have been or are They are generated of living and are available to replace animal immunisation for a range of are selected in vitro by or These are but also affinity reagents such as and [6Taussig M.J. et al.ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome.Nat. Methods. 2007; 4: 13-17Crossref PubMed Scopus (210) Google Scholar, S. et recombinant antibodies to the human Biotechnol. PubMed Scopus Google Scholar]. 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