Animal-derived antibodies are a major contributor to the reproducibility crisis in research.1 They often show poor specificity or fail to recognise their targets. In a systematic analysis of 185 commercially available hybridoma monoclonal antibodies, one-third were not reliably monospecific,2 and only 0.5% to 5% of the antibodies in a polyclonal reagent bind to their intended target.3 In a 2015 Nature commentary, 111 academic and industry scientists called for an international shift to the use of recombinant antibodies (rAbs) for reasons that include increased reliability and reduced lot-to-lot variability in affinity reagents.3
The use of faulty antibodies can cost laboratories thousands of dollars a year and “trigger junk-data avalanches through the literature”.4 Select publications describing how undefined, animal-derived antibodies have inhibited progress in identifying and treating disease include the following:
Vaezi et al. reported that an animal-derived monoclonal antibody used in a majority of studies to measure an antigen previously thought to be predictive of treatment outcome for non‐small cell lung cancer was not monospecific and that another target indicated clinical outcome.5
Andersson et al. found that antibodies used over the past 20 years did not specifically target oestrogen receptor β (ERβ), a historically important target in breast cancer research, and that ERβ protein is not found in breast tissue.6
Gilda et al. showed that five commercial ubiquitin antibodies resulted in different detection of free ubiquitin or ubiquitinated proteins in western blot analysis. Furthermore, large variability was seen in the ability of six commercial antibodies to detect a protein modifier.7
Elliott et al. found that anti-erythropoietin receptor (EpoR) antibodies were not specific in immunohistochemical methods, resulting in unreliable results from cancer studies reporting EpoR expression.8
Baker recounts how years of research and millions of dollars were wasted on a cancer treatment that relied on antibodies that proved to be unreliable.1
The need for higher-quality affinity reagents has prompted the development of sequence-defined, animal-free rAbs. Their advantages include the following:
Antibody selection conditions can be adjusted to select for a specific epitope structure, antibody conformation, and cross-reactivity.9,10
Negative selection techniques can exclude antibodies that react with molecules similar to the selected antigen.10
Because they are independent of a biological immune response, antibodies can be developed to target toxic and pathogenic antigens.9,11
Antibodies can be developed to target molecules with low molecular weight, such as haptens, that fail to elicit an immune response in animals.12 They can also be produced to target peptides and non-biological targets, including metals and carbon nanotubes.13
They are available in multiple sizes – from fragments to full-length antibodies.9
Their sequence is known and can be reformatted.14 Because the sequence is known, the same antibody can be resynthesized when needed.9
They are broadly applicable and can be used in immunohistochemistry, western blot, ELISA, flow cytometry, and immunoprecipitation.9
Because they are already of human origin, they do not have to be humanised for therapeutic use.9
For more information, see Resources.
While costs are similar for previously produced “catalogue” animal-derived antibodies and rAbs, the initial investment in new, custom-made antibodies is high, whether they are generated using animals or non-animal methods.9 Because animal-derived antibodies have been produced for decades, many do not have to be custom-made, whereas many rAbs are not yet available in catalogues and so must be custom-made. Therefore, initially, the production of new rAbs can be expensive, as is the case for many forms of new technology, but the price should decrease as more companies and universities become involved in their development and use.
Considerable cost savings are associated with the more reproducible research that will result from using higher-quality, animal-free rAbs. Bradbury and Plückthun estimate that US$800 million is wasted annually worldwide on unreliable antibodies, US$350 million of that in the US.2Practical Reasons
Non-animal affinity reagents are faster to make than animal-derived antibodies. Once an antibody library is established, it takes approximately two to eight weeks to produce a recombinant antibody. In contrast, it takes four or more months to produce an antibody using animals.11
Furthermore, rAbs are cheaper to handle, transport, and store.9 Polyclonal antibody serum must be kept frozen and requires significant freezer space. Hybridoma cells for monoclonal antibodies must be kept frozen in liquid nitrogen, and each hybridoma cell line must be thawed and refrozen every few years and occasionally recloned and retested.14 On the other hand, the gene encoding a given rAb can be stored in the form of highly stable plasmid DNA or in an in silico database.14Ethical Reasons
Millions of animals are used to produce antibodies every year. Animals used in antibody production are subjected to invasive and painful procedures. Furthermore, numerous animal welfare violations have been uncovered at facilities that produce antibodies.
In the ascites method of monoclonal antibody production, tumour development, accumulation of ascites fluid, and multiple fluid draws can cause the animals, usually mice, considerable pain and distress. It has been reported that they are unable to eat, walk, or breathe properly. A number of countries, such as Australia, Canada, Germany, the Netherlands, Switzerland, and the UK, have restricted or banned the production of antibodies via the ascites method because of animal welfare concerns.15–17
Photographs of rabbits at the antibody-production facilities ProSci Inc., Southern Biotechnology Associates Inc., Colorado Serum Company, Novus Biologicals, and Robert Sargent were obtained by PETA in response to Freedom of Information Act requests.
Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015;521(7552):274-276. doi:10.1038/521274a
Bradbury ARM, Trinklein ND, Thie H, et al. When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. MAbs. 2018;10(4):539-546. doi:10.1080/19420862.2018.1445456
Bradbury A, Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518(7537):27-29. doi:10.1038/518027a
Goodman SL. The path to VICTORy – a beginner’s guide to success using commercial research antibodies. J Cell Sci. 2018;131(10). doi:10.1242/jcs.216416
Vaezi AE, Bepler G, Bhagwat NR, et al. Choline phosphate cytidylyltransferase-α is a novel antigen detected by the anti-ERCC1 antibody 8F1 with biomarker value in patients with lung and head and neck squamous cell carcinomas. Cancer. 2014;120(12):1898-907. doi:10.1002/cncr.28643.
Andersson S, Sundberg M, Pristovsek N, et al. Insufficient antibody validation challenges oestrogen receptor beta research. Nat Commun. 2017;8. doi:10.1038/ncomms15840
Gilda JE, Ghosh R, Cheah JX, et al. Western blotting inaccuracies with unverified antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS). PLoS ONE. 2015;10(8): e0135392. doi:10.1371/journal.pone.0135392
Elliott S, Busse L, Bass MB, et al. Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood. 2006;107(5):1892-1895. doi:10.1182/blood-2005-10-4066
Dübel S, Stoevesandt O, Taussig MJ, Hust M. Generating recombinant antibodies to the complete human proteome. Trends Biotechnol. 2010;28(7):333-339. doi:10.1016/j.tibtech.2010.05.001
Groff K, Brown J, Clippinger AJ. Modern affinity reagents: Recombinant antibodies and aptamers. Biotechnol Adv. 2015;33(8):1787-1798. doi:10.1016/j.biotechadv.2015.10.004
Ferrari D, Garrapa V, Locatelli M, Bolchi A. A novel nanobody scaffold optimized for bacterial expression and suitable for the construction of ribosome display libraries. Mol Biotechnol. 2020;62(1):43-55. doi:10.1007/s12033-019-00224-z
Kavanagh O, Elliott CT, Campbell K. Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins. Anal Bioanal Chem. 2015;407(10):2749-2770. doi:10.1007/s00216-015-8502-z
Soshee A, Zürcher S, Spencer ND, Halperin A, Nizak C. General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules. 2014;15(1):113-121. doi:10.1021/bm401360y
Cosson P, Hartley O. Recombinant antibodies for academia: A practical approach. Chimia (Aarau). 2016;70(12):893-897. doi:10.2533/chimia.2016.893
Hendriksen CFM. Replacement, reduction and refinement in the production and quality control of immunobiologicals. AATEX. 2006;11(3):155-161. doi:10.11232/aatex.11.155
Marx U, Embleton MJ, Fischer R, et al. Monoclonal antibody production: The report and recommendations of ECVAM workshop 23. Altern Lab Anim. 1997;25(2):121-137.
Clark A, Befus D, O’Hashi P, et al. Canadian Council on Animal Care guidelines on: Antibody production. 2002. www.ccac.ca/Documents/Standards/Guidelines/Antibody_production.pdf.