Replacing animal-derived antibodies with animal-free affinity reagents

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Antibodies in research

In vitro antibody production and animal-free antibody technologies offer reagents that provide scientific and 3Rs advantages over animal-derived products [1]. Despite this, there has been little shift towards using them in common research practice. This resource provides guidance on animal-free antibody technologies and their adoption.

Animal-derived antibody production typically involves immunising between one and three animals (usually mice or rabbits, but sometimes sheep, chickens, goats or donkeys) per target. Multiple attempts are often needed to generate antibodies for certain targets, further increasing animal use. An estimated one million animals are used per year in the EU alone for antibody production [1].

Demand for antibodies continues to rise, with the global research antibodies market size estimated to reach $5.6 billion USD by 2027 [2]. However, animal-derived antibodies are expensive and prone to instability during storage, have variable specificity, and can suffer from batch-to-batch variation, adversely affecting the reproducibility of research [3]. The loss in time and resources from poorly characterised antibodies is estimated at $800 million USD per year [4].

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Animal-free antibody technologies

There are two main types of animal-free antibody technologies:

  • Non-animal-derived antibodies: antibodies developed using in vitro recombinant libraries that do not use animal immunisation during their generation or production.
     
  • Affinity reagents: synthetically derived polypeptides or oligonucleotides that bind to defined, specific targets with high affinity comparable to animal-derived antibodies.

Non-animal-derived antibodies and affinity reagents are mature technologies that are commercially available, are amenable to most research applications and offer significant scientific benefits (summarised in Table 1). This is supported by a recent review from the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) [1] which recommends that animals should no longer be used for the development and production of antibodies for research.

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Benefits of animal-free antibody technologies

Specificity – epitope, conformation and cross-reactivity are defined during the production process and cross-reactivity can be eliminated. 

Design – can be designed for a broader range of targets, for example, human targets conserved across species, small molecules and those that are non-immunogenic. 

Stability – many non-animal technologies are stable through wider pH ranges, temperatures and in various solvents.

Unlimited supply – as the sequence is known, there is no limit to, or variation of, the antibodies produced.

Faster production – recombinant antibodies from established libraries can be produced in a matter of weeks.

Affinity – through stringent selection and optimization to user specific protocols, higher affinities can be achieved in all well-known molecular formats.

Cost – production costs are similar to those of monoclonal antibodies generated with animals, and the increase in specificity and reproducibility offsets the increased cost over polyclonal antibodies. 

Broad applicability – can be used for the majority of bench applications.

Translatability – because non-animal technologies are of human origin, they do not have to be humanized for therapeutic use.

Table 1: The benefits of using animal-free antibody technologies.

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Adopting animal-free antibody technologies

Animal-free antibodies suitable for research applications are available from suppliers and through custom generation services (see Resources). However, misconceptions of their validity and limited awareness of their advantages [1] are preventing widespread uptake of these technologies within the research community.

Supporting the adoption of animal-free antibody technologies will require the combined efforts of manufacturers, suppliers and end-users. Further guidance for the adoption of animal-free antibody technologies can be found in the EURL ECVAM Recommendation on Non-Animal-Derived Antibodies review [1].

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Case studies

We have funded and showcased projects developing animal-free antibody technologies.

Affimers

 

Affimers are engineered binding proteins that possess the desirable properties of antibodies such as high specificity and affinity, whilst avoiding some of the problems commonly associated with antibody use such as cross-reactivity and fragility. They have similar binding affinities as antibodies and are stable when bound to surfaces. These properties make them ideal for applications that require immobilisation of a capture reagent, as well as use in a variety of assays that have traditionally used antibodies. Custom Affimers are generated through in vitro selection in a fraction of the time it takes to develop a new antibody and can even be made in cases where it is impossible to raise antibodies: targets do not need to be immunogenic, and toxicity to the host is not an issue.

 

Affimers have been shown to be effective in common molecular and cellular applications [5], imaging the actin cytoskeleton [6] and potentially for therapeutic applications in autoimmunity, via competitive or allosteric modes of action [7].

 

We are funding a research project to employ Affimer technology for secondary antibody applications. The researchers, based at the University of Leeds, aim to replace secondary antibodies produced in mammals with Affimers labelled with fluorophores or horseradish peroxidase, and compare their efficacy with commercially available secondary antibodies. Successful implementation of the technology could have a significant impact on the number of animals used for secondary antibody production.

Molecularly imprinted polymers

 

Polymers, or ‘plastic’ antibodies, can mimic the properties of natural antibodies. These synthetic antibodies, referred to as molecularly imprinted polymers (MIPs), are made by polymerising vinylic monomers in the presence of a specific target [8]. After removal of the target, a porous structure is obtained with imprints that have a high specificity towards the target. MIPs are an emerging technology that are finding their first commercial applications; chromatography columns packed with MIP particles used for extraction can be obtained from companies such as Biotage and Sigma Aldrich [9].

 

Advantages of MIPs over antibodies include superior stability – MIPs can operate at up to 100°C and are stable in organic solvents and at extreme pH levels. They are prepared in bulk quantities and give consistent results, contrary to the batch-to-batch variability frequently observed with antibodies. Molecular imprinting technology is versatile, and it is possible to tailor the polymer for specific targets, meaning they can be utilised to detect a wide range of biological targets from small ions to larger proteins.

 

Through our Innovation Platform, researchers at Manchester Metropolitan University were able to validate a novel biosensor platform using MIPs, combining electrochemical detection and a novel thermal technique, to detect cardiac biomarkers. We have also funded researchers applying MIP technology for therapeutic and diagnostic purposes. Dr Allesandro Poma has developed molecularly imprinted nanoparticles (nanoMIPs) that can be drug-loaded for targeted therapeutics and are a plausible alternative to conventional antibodies.

Aptamers

 

Aptamers are short, synthetic sequences of single-stranded oligonucleotides that undergo in vitro selection on the basis of their binding or catalytic activity (termed SELEX). They are selected from an ‘initial oligonucleotide pool’, comprised of random sequences. The SELEX process was developed about 30 years ago, and since then researchers have identified numerous aptamers targeting a broad range of proteins and have continued to advance the SELEX methodology10. For example, live cells are used to select oligonucleotides in a process termed Cell-SELEX, an approach that has useful applications in oncology.

 

They can be deployed in every application for which an antibody might be used, and are relatively inexpensive, non-immunogenic and can be easily modified11. Aptamers have also been developed in diagnostic and therapeutic settings. There are examples of aptamers that have entered clinical trials in a range of contexts, such as cancer, ocular disease and inflammation. Due to their versatility and animal-free origin, aptamers are a promising molecular tool with a broad range of potential applications.

 

We have previously showcased a project by Aptamer Group, who have successfully developed next-generation aptamer molecules termed Optimers, an animal-free alternative to antibodies with diagnostic and therapeutic applications.

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Resources

Below is a non-exhaustive list of companies supplying animal-free antibody technologies, and we recommend that researchers discuss animal-free options with their usual suppliers.

Companies supplying non-animal-derived recombinant antibodies:

Organisations and companies offering custom generation of non-animal-derived antibodies:

Organisations and companies offering phage display antibody library construction from non-animal sources. Libraries may be limited to research purposes or available for out-licensing to the biotechnology and pharmaceutical community:

Other affinity reagents:

If you are a company or institution offering animal-free antibody technologies and would like to be added to the resource list, please get in touch.

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Further reading

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References

  1. Viegas Barroso JF, Halder ME and Whelan M (2020). EURL ECVAM Recommendation on Non-Animal-Derived Antibodies, EUR 30185 EN. Publications Office of the European Union: Luxembourg.
     
  2. Emergen Research (2020). Research Antibodies Market By Market By Product, By Antibody Type (Monoclonal, Polyclonal), By Technology, By Application, By End-Users (Pharmaceutical & Biopharmaceutical Firms, Academic & Research Institutes, Contract Research Organizations), Forecasts to 2027.
     
  3. Kusnezow W and Hoheisel JD (2002). Antibody microarrays: promises and problems. Biotechniques Supplement 14-23. PMID: 12514925
     
  4. Baker M (2015). Antibody anarchy: A call to order. Nature 527(7579): 545–551. doi: 10.1038/527545a
     
  5. Tiede C et al. (2017). Affimer proteins are versatile and renewable affinity reagents. eLife 6:e24903. doi: 10.7554/eLife.24903
     
  6. Lopata A et al. (2018). Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells. Scientific Reports 8(6572). doi: 10.1038/s41598-018-24953-4
     
  7. Robinson JI et al. (2018). Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action. PNAS 115(1): e72–81. doi: 10.1073/pnas.1707856115
     
  8. Haupt K and Mosbach K (2000). Molecularly Imprinted Polymers and Their Use in Biomimetic Sensors. Chemical Reviews 100(7): 2495–2504. doi: 10.1021/cr990099w
     
  9. Nestora S et al. (2016). Plastic Antibodies for Cosmetics: Molecularly Imprinted Polymers Scavenge Precursors of Malodors. Angewandte Chemie International Edition 55(21): 6252–6256. doi: 10.1002/anie.201602076
     
  10. Keefe AD, Pai S and Ellington A (2010). Aptamers as therapeutics. Nature Reviews Drug Discovery 9: 537–550. doi: 10.1038/nrd3141
     
  11. Lakhin AV, Tarantul VZ and Gening LV (2013). Aptamers: Problems, Solutions and Prospects. Acta Naturae 5(4): 34–43. PMID: 24455181

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