Improving Research Reproducibility and Animal Welfare
by Replacing Animal-Derived Antibodies
with Non-Animal Affinity Reagents
CONTENTS
Introduction _____________________________________________________________1
Animal-Derived Antibodies
About ________________________________________________________________1
Scientific Issues ________________________________________________________2
Animal Welfare Issues ___________________________________________________3
Economic Issues ______________________________________________________10
Non-Animal Affinity Reagents
About _______________________________________________________________10
Scientific Benefits _____________________________________________________12
Barriers and the Path Forward _____________________________________________12
INTRODUCTION
Affinity reagents, such as antibodies, are essential tools used in research to bind to a molecule to
identify it or influence its activity. However, researchers report growing concern about the lack
of quality and reproducibility of animal-derived antibodies, which often show poor specificity or
fail to recognize their targets.1,2,3 As a result, an important step to improve research
reproducibility is to replace animal-derived antibodies with non-animal affinity reagents.
Non-animal affinity reagents, including recombinant antibodies and aptamers, can be used in all
applications in which traditional antibodies are used.4 This report describes how time, money,
and animals could be saved, while improving the reproducibility of research, by replacing
animal-derived antibodies with modern technologies.
ANIMAL-DERIVED ANTIBODIES
About
Both monoclonal and polyclonal antibodies are used in research. Monoclonal antibodies are
derived from a single line of antibody-producing cells and, therefore, selectively bind to a single
epitope on an antigen. There are two general ways that monoclonal antibodies are produced
using animals – the ascites and “in vitro” methods – which share the same initial development
steps, as indicated in the figure below. These methods entail immunizing an animal with an
antigen (often multiple times over a period of weeks), killing the animal, dissecting the spleen to
isolate antibody-producing cells, and fusing the antibody-producing cells with immortal tumor
cells. The next steps can occur either in vivo (ascites method) or in vitro (“in vitro” method). In
1

Bradbury A, Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518(7537):27-29.
Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015;521(7552):274-276.
3
Berglund L, Björling E, Oksvold P, et al. A genecentric Human Protein Atlas for expression profiles based on
antibodies. Mol Cell Proteomics. 2008;7(10):2019-2027.
4
Groff K, Brown J, Clippinger AJ. Modern affinity reagents: Recombinant antibodies and aptamers. Biotechnol Adv.
2015;33(8):1787-1798.
2

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 the ascites method, the fused cells (hybridomas) are injected into the abdomens of mice, where
they multiply and produce antibody-containing fluid (ascites). Tumor development,
accumulation of ascites fluid, and multiple fluid draws can cause the mice considerable pain and
distress. In the “in vitro” method, a refinement compared to the ascites method, hybridomas are
cultured and multiplied in vitro in flasks or bioreactors.5

Methods of monoclonal antibody production in animals
Polyclonal antibodies are derived from multiple lines of antibody-producing cells and, therefore,
recognize multiple epitopes on an antigen. Polyclonal antibodies are produced by immunizing an
animal and then harvesting antibodies from the animal’s serum. Larger animals are often used
for the production of polyclonal antibodies because a larger amount of serum can be collected.
Scientific Issues
Commercial antibodies often show poor specificity or fail to recognize their targets. In a
February 2015 Nature commentary, 109 academic and industry scientists joined Andrew
Bradbury of the Los Alamos National Laboratory and Andreas Plückthun, head of the
Department of Biochemistry at the University of Zurich, to call for an international shift to the
use of recombinant antibodies for reasons that include increased reliability and reduced lot-to-lot
variability in affinity reagents.6 Bradbury and Plückthun note that they believe that poorly
characterized and ill-defined antibodies were in large part to blame in a study in which the
5
6

For more information and citations, see: Groff et al.
Bradbury, Plückthun.

2

 scientific results of only six of 53 landmark preclinical studies could be replicated. In addition,
because only 0.5 to 5 per cent of the antibodies in a polyclonal reagent bind to their intended
target and polyclonal reagents have significant batch-to-batch variation, the scientists called for
polyclonal antibodies to be phased out of research completely.
Additional publications have highlighted the lack of reliability of traditional antibodies. A 2015
Nature news feature reports that antibodies may be the laboratory tool most commonly
contributing to the “reproducibility crisis”,7 and a systematic analysis of 185 commercially
available hybridoma monoclonal antibodies published in 2018 found that one-third were not
reliably monospecific. The authors recommended the transition to sequence-defined recombinant
antibodies.8
Animal Welfare Issues
Tens to hundreds of thousands of animals, including mice, rats, rabbits, llamas, camels, goats,
sheep, horses, and other animals, are used in the production of affinity reagents every year.
Animals used in antibody production are subjected to invasive and painful procedures. In the
ascites method of monoclonal antibody production, tumor development, accumulation of ascites
fluid, and multiple withdraws of fluid can cause the animals, usually mice, considerable pain and
distress. Animals have been reported to be unable to eat, walk, or breathe properly. A number of
countries, such as Australia, Canada, Germany, the Netherlands, Switzerland, and the United
Kingdom, have restricted or banned the production of antibodies via the ascites method because
of animal welfare concerns.9 In the US, while mice are commonly used in monoclonal antibody
production, they are not covered by the Animal Welfare Act (AWA). Therefore, their well-being
and conditions are not inspected by the United States Department of Agriculture (USDA).
Numerous animal welfare violations have been uncovered at facilities that produce antibodies.
For example, the USDA filed three complaints against Santa Cruz Biotechnology (SCBT) – on
19 July 2012, 4 November 2014, and 7 August 2015. The agency cited SCBT for approximately
40 violations of the AWA within five years, including the following:




7

Neglecting to provide a goat who had a complete break in one of his legs with continued
veterinary care. When USDA inspectors visited, the lower portion of the leg was moving
independently.
Failing to treat a goat who lost a quarter of his bodyweight because he had a swollen jaw and
draining lesion from a rattlesnake bite to the face and was unable to eat
Failing to treat a severely ill goat, who died during the USDA inspection. The inspectors
noted that the goat could most likely have been successfully treated with antibiotics if the
condition had been identified and treated in a timely manner.

Baker.
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.
9
Groff et al.
8

3

 Other facilities, including Pi Bioscientific, Novus Biologicals, and ProSci Inc, have each been
cited for multiple AWA violations in the past few years:





Pi Bioscientific was cited for failing to provide dozens of goats who suffered from
emaciation, diarrhea, lameness, overgrown hooves, and/or upper respiratory disease with
adequate care, among other violations. The company was cited for 20 violations in just six
days in March 2016, and it has continued to violate the AWA. In March 2018, it was cited
for seven violations, including failing to provide at least eight suffering goats and sheep with
veterinary care – and continuing to deny at least four of those animals care for three weeks
after being cited.10
March 2018 citations at Novus Biologicals include severe weight loss in rabbits, with two
rabbits being found dead in their cages. These issues were not reported to a veterinarian.11
Federal inspectors have documented more than 35 violations of animal welfare laws at
ProSci Inc over the last three years. Rabbits were confined to small, rusted, filth-encrusted
wire cages without even a resting board. Some had painful eye infections with discharge
exuding from their eyes. Others suffered from head tilt – which can be painful and prevent
them from eating and drinking – as well as parasites, overgrown nails, excessive amounts of
dried blood on their ears and/or backs, and emaciation. Goats had overgrown and cracked
hooves, llamas had excessively long hooves, and cows and sheep had inadequate shelter. The
USDA records also reveal mold and rat faeces in the animals’ food, deep cracks filled with
waste in the cement flooring, and rust, debris, and excrement on the cages housing rabbits.12

The photos below were taken by USDA inspectors at Pi Bioscientific, Novus Biologicals, ProSci
Inc, and SCBT.13 They reveal that animals are held in bleak conditions, deprived of basic care,
and left to suffer from untreated infections.

10

For additional photographs, see: www.piscltd.org.uk/wp-content/uploads/2018/08/2018.08.06-Pi-Bioscientificphotos.pdf.
11
For additional photographs, see: www.piscltd.org.uk/wp-content/uploads/2018/08/2018.07.19-Novus-BiologicalsFOIA-response.pdf.
12
For additional photographs, see: www.piscltd.org.uk/wp-content/uploads/2018/08/2017-APHIS-02031-FReviewed-Records-93-R-0521-full-release.pdf and www.piscltd.org.uk/wp-content/uploads/2018/08/2017-APHIS02031-F-Reviewed-Records-93-B-0229-full-release.pdf.
13
Photos were obtained by PETA US via Freedom of Information Act requests.

4

 Sheep held in filthy, wet conditions at Pi Bioscientific in March 2016

An emaciated and coughing goat with overgrown hooves and mucoid discharge at
Pi Bioscientific in March 2016 (left). An emaciated goat with diarrhea at
Pi Bioscientific in March 2016 (right)

5

 Close-up of a goat’s twisted and severely overgrown hoof, at Pi Bioscientific in March 2016
(left). A goat’s overgrown hoof, at Pi Bioscientific in March 2016 (right)

Rabbits with head tilts who were in need of veterinary care at ProSci Inc in July 2015

A rabbit with thick discharge in his or her eye (left) and an underweight, depressed, and
soiled rabbit in need of veterinary care, according to USDA documentation (right),
at ProSci Inc in July 2015

6

 Rabbits kept in barren wire cages, some of which do not meet minimum space requirements,
at ProSci Inc in March 2016 (left). Rabbits kept in dirty, barren wire cages at
Novus Biologicals in March 2018 (right).

A rabbit with untrimmed toenails kept in a dirty and barren wire cage at Novus Biologicals
in March 2018 (left) and a close-up of a rabbit’s overgrown toenails at
ProSci Inc in July 2015 (right)

7

 Sheep kept in a muddy pen with insufficient shelter to protect them all from the rain (left)
and sheep with only a cloth shade for cover and no protection from the rain (right)
at ProSci Inc in September 2016

A goat with a tumour on his or her neck (left) and a goat with a wound
on his or her leg (right) at SCBT14
Furthermore, the conditions of animals used in antibody production outside the US whose
antibodies are then imported into the US are not inspected by the USDA. For example, almost all
commercial antitoxins (antibodies that bind to toxins) have been manufactured using serum from
equines who have been hyperimmunised by repeated toxin injections. In 2015, inspections of
equine-serum production facilities in India found substandard living conditions, a lack of
appropriate veterinary care, and routine negligence of basic animal welfare laws. In addition to
painful complications caused by being repeatedly injected with toxins and then bled, the animals
showed signs of lameness, diseased hooves, eye abnormalities, and malnutrition. These facilities
did not provide humane euthanasia to animals who were too ill to recover from advanced illness
or injury.15
14

Hart P. Santa Cruz Biotechnology agrees to record fine to settle animal abuse charges. Huffington Post, 21 May
2016.
15
Inspections were conducted at 10 facilities by veterinarians and other experts from veterinary colleges and nongovernmental organizations, including PETA India. Inspection footage can be found at PISCLtd.org.uk/antitoxin.

8

 Horses with wounds and kept in substandard living conditions at antibody-production facilities
in India. Photos: PETA India

9

 Economic Issues
Considerable cost savings are associated with the more reproducible research that will result
from using higher-quality antibodies. Bradbury and Plückthun estimate that US$800 million is
wasted annually worldwide on unreliable antibodies, US$350 million of that in the US.16 For
example, Baker recounts the story of David Rimm, a pathologist at Yale University, who spent
years of research and millions of dollars on a cancer treatment that relied on antibodies that
proved to be unreliable.17
The high affinity and specificity of non-animal affinity reagents leads to cost savings through
more reproducible research. In addition, non-animal affinity reagents are much faster to make
than animal-derived antibodies. Aptamers can be synthesized within days, and 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.18
NON-ANIMAL AFFINITY REAGENTS
About
To increase research reliability and reproducibility and minimize animal use, researchers should
use recombinant antibodies (rAbs) or other non-animal affinity reagents, such as aptamers.
Recombinant antibodies are protein-based reagents that are selected from a library of genes
encoding slightly different antibody proteins for their affinity to bind to target antigens.
Aptamers are short, single-stranded DNA or RNA oligonucleotides that detect, characterize,
and/or modify the activity of their targets. Detailed information on the production processes for
rAbs and aptamers can be found in “Modern Affinity Regents: Recombinant Antibodies and
Aptamers”.19,20
Developed by researchers in their laboratories and commercially available, rAbs and aptamers
can be used in all applications in which traditional antibodies are used, including in basic
research, regulatory testing, and clinical applications.

16

Bradbury, Plückthun.
Baker.
18
For more information and citations, see: Groff et al.
19
Ibid.
20
See also: Bradbury ARM, Sidhu S, Dübel S, McCafferty J. Beyond natural antibodies: The power of in vitro
display technologies. Nat Biotechnol. 2011;29(3):245-254; Unkauf T, Miethe S, Fühner V, Schirrmann T, Frenzel
A, Hust M. Generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and
therapy. Adv Exp Med Biol. 2016;917:55-76; Frenzel A, Schirrmann T, Hust M. Phage display-derived human
antibodies in clinical development and therapy. mAbs. 2016;8(7):1177-1194.
17

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 Basic Research




Identification and detection of the concentration of
molecules, biological compounds, viruses, residues in
food, or diseased cells by conjugation with peptide tags,
proteins, or nanoparticles that give them fluorescent
properties
Commonly performed assays, such as
immunofluorescence, microscopy, microarrays,
immunocytochemistry, immunohistochemistry, flow
cytometry, ELISA, and blotting assays

Regulatory Testing


Safety and efficacy testing for regulatory purposes (e.g., in
an assay to determine vaccine potency, in quality-control
testing of therapeutic proteins, or in the detection of
environmental contaminants)



Imaging, including for treatment monitoring and cancer
detection
Therapeutics, by altering target activity or by delivery of
therapeutic agents to target cells via conjugation to
antibiotics, RNA interference, toxins, enzymes, or drugs
Antitoxins

Clinical Applications



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 Scientific Benefits
Recombinant antibodies and aptamers have numerous scientific advantages over animal-derived
antibodies, including the following:









They can be produced more rapidly.
They can be produced with higher affinity levels and binding specificity.
They can exhibit greater versatility, allowing, for example, the ability to generate antibodies
and aptamers against toxic or non-immunogenic antigens.
Batch-to-batch variation is limited because the sequences are known and their generation
does not rely on biological processes.
They can detect smaller amounts of antigen.
Production can be carried out under controlled selection conditions.
Compared to animal-derived antibodies, they elicit a reduced immune response when used in
therapeutic applications because they are free of animal contaminants.
They can be made without using animals.21,22

These advantages are described in more detail in “Modern Affinity Regents: Recombinant
Antibodies and Aptamers”.23
BARRIERS AND THE PATH FORWARD
While there are a handful of factors that have slowed the adoption of recombinant antibodies and
other non-animal affinity reagents, all the barriers can be overcome and are less concerning than
the use of unreliable and irreproducible animal-derived antibodies. From a scientific and animal
welfare perspective, it is clear that there is a need for more reliable, specific, and versatile nonanimal affinity reagents, and government agencies should play a key role in their uptake.
One barrier has been a lack of awareness among the research community of the benefits
associated with recombinant antibody and aptamer technologies and a reliance on outdated
information. To address this issue, the US National Institutes of Health (NIH) should
commission an updated review of antibodies and other affinity reagents. In 1999, the
National Research Council (NRC) published an NIH-commissioned study evaluating whether
there was a scientific necessity to use animals to produce monoclonal antibodies.24 Even though
the report, “Monoclonal Antibody Production”, did not include information about rAbs,
aptamers, or other non-animal affinity reagents, NIH’s Public Health Service (PHS) Policy on
Humane Care and Use of Laboratory Animals still relies on the report to set its policy on the
acceptability of the ascites method.25 In the nearly 20 years since the report was published,
significant advances have been made in rAb and aptamer technologies, highlighting the need for

21

Gray AC, Sidhu SS, Chandrasekera PC, Hendriksen CFM, Borrebaeck CAK. Animal-friendly affinity reagents:
Replacing the needless in the haystack. Trends Biotechnol. 2016;34(12):960-969.
22
Groff et al.
23
Ibid.
24
National Research Council. Monoclonal antibody production. Report of the Committee on Methods for Producing
Monoclonal Antibodies. National Academy Press, Washington, DC, 1999.
25
US National Institutes of Health. PHS policy on humane care and use of laboratory animals: Frequently asked
questions. Revised 28 November 2017. Available at: https://olaw.nih.gov/guidance/faqs.

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 an updated report that includes an evaluation of monoclonal and polyclonal antibodies (including
antitoxins).
Cost has been another major barrier to the adoption of rAbs. While costs are similar for
previously produced “catalog” animal-derived antibodies and rAbs, the initial investment in new,
custom-made antibodies is high. 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
catalogs and so must be custom-made. Therefore, initially, the production of new rAbs are often
expensive, as are many forms of new technology, but the price should decrease as more
universities and companies become involved in their development and use. For this reason, it is
important that funding from the federal government is available for researchers interested in the
development and use of non-animal affinity reagents. Specifically, the NIH should join with
industry, academia, and non-governmental organizations to identify which commonly used
affinity reagents to prioritize for development, and NIH funding should be made available
for researchers interested in the development, use, and validation of recombinant
antibodies or aptamers.
Non-animal affinity reagents are scientifically and ethically superior laboratory tools. They are
the way forward to increased scientific validity and reproducibility and to accelerated research in
the life sciences. For more information, please see PISCLtd.org.uk/antibodies.

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