Take a look at the picture below. They are
immunohistochemistry experiments in which three antibodies (green) are directed
towards the same mitochondrial protein. Each picture represents one of the
three antibodies. The unexpected pattern on the picture to the right shows that
the third antibody binds an unintended protein. Clearly! But how can this
happen? How can a commercially available antibody not only NOT bind to its
supposed target but bind to an ENTIRELY DIFFERENT one?
The saddest lab meeting
An antibody that performs differently across
experiments can cause calamity. Examples are endless and one of them is the
case of David Rimm a pathologist at Yale
University in New Haven, USA.
In 2006, things were looking pretty good for David, he had
developed a test to guide effective treatment of the skin cancer melanoma and
it promised to save lives. He had found a combination of antibodies that, when
used to ‘stain’ tumor biopsies, produced a pattern that indicated whether the
patient would need to take certain harsh drugs to prevent a relapse after
surgery. He had secured more than US$2 million in funding to move the test
towards the clinic. But in 2009, everything started to fall apart. When he
ordered a fresh set of antibodies, his team could not reproduce the original
results. The antibodies were sold by the same companies as the original batches
and were supposed to be identical — but they did not yield the same
staining patterns, even on the same tumors. Rimm was forced to give up his work
on the melanoma antibody set. In his own words, “That was a very sad lab
meeting” (1).
There are signs that problems with antibodies are having
broad and potentially devastating effects on the research record and I’m pretty
sure that almost every scientist that has worked with antibodies has had some
kind of ‘issue’ with them, but it has been difficult to gauge the size of the
problem across biology as a whole.
Ugly statistics
The Human Protein Atlas, a Swedish consortium that aims to
generate antibodies for every protein in the human genome, has looked at some
20,000 commercial antibodies so far and found that less than 50% can be used
effectively to look at protein distribution in preserved slices of tissue (2),
in other words, up to half of all
commercially available antibodies are unreliable for that. Now that’s a
shocking number.
Some other ugly numbers come from epigenetics. And I can
tell you from the inside that this field relies heavily on antibodies, that’s a
fact. In 2011, an evaluation of 246 antibodies used in epigenetic studies found
that 25% failed tests for specificity, meaning that they often bound to more
than one target (3). Four antibodies
were perfectly specific — but to the wrong target!!!!!!!!!
In 2012, a group of Amgen researchers
attempted to reproduce the results of 53 “landmark” cancer papers; only 6 had
scientific findings that could be replicated (4). In several instances, the
analysis found that failure to reproduce experimental data could be attributed
to antibodies that were nonspecific or poorly validated. This lack of
reproducibility can also spill over into ineffective diagnostics that delay
clinical research.
Who’s to blame?
Should we blame it on antibodies? Of course not. We are not talking about good and bad
antibodies, but antibodies that work in specific assays and specific context. Then, should we blame it on the bossanova or what? Who is
responsible for such despair results? I would dare to say that companies that
produce antibodies are the main responsibles, but buyers and people that handle
them play a good part in that too. I’ll dig into this:
Antibodies are ubiquitous tools in the
life sciences with a market for research antibodies at US$2.5 billion a year (ironically, losses
from purchasing poorly characterized antibodies have been estimated at $800 million per year, not counting the
impact of false conclusions, uninterpretable or misinterpreted experiments,
wasted patient samples and fruitless research time). But besides being a good
business, no big efforts have been done to characterize them
at least until this whole antibody worldwide rebellion against antibodies started.
There are
no uniform or enforceable standards for antibody validation. Unlike drugs, there
is no agency governing what can be sold into the antibody-based assay
market.
Most scientists who purchase antibodies
believe the label printed on the vial. But believe it or not, we were buying
non-validated antibodies all the way. Take the example of Ioannis Prassas, a
proteomics researcher at Mount Sinai Hospital in Canada. He and his colleagues
had been chasing a protein called CUZD1, which they thought could be used to
test whether someone has pancreatic cancer. They bought a protein-detection kit
and wasted two years, $500,000 and thousands of patient samples before they
realized that the antibody in the kit was recognizing a different cancer protein, CA125, and did not bind to CUZD1 at all (5).
Unbelievable!
So YES,
scientists need to be more carefull. Be sure of what you have between your
hands and what it has been made for. Antibodies are not magic reagents. You
can't just throw them on your sample and expect a result, when for example many
companies explicitly states the types of experiment that an antibody should be
used for. An antibody might work great in western blots but not at all in
immunohistochemistry. Of course many scientists do not always follow the
instructions.
A few scientists have begun to
speak up. David Rimm's disappointment set him on a crusade to educate others by
writing reviews, hosting web seminars and raising the problem in countless
conference talks. He and others are calling for the creation of standards by
which antibodies should be made, used and described.
In September 2015, the International Working Group on Antibody Validation, a
group of leading authorities in the field of protein-binding technology, had
its first meeting in Canada. The goal: to develop
common validation standards for antibodies. That same month, the Federation of American
Societies for Experimental Biology (FASEB) hosted roundtables to explore
problems with antibodies. It expects to issue recommendations early this year.
The US
National Institutes of Health (NIH) is also on the case. They recently (Jan
2016) included a new section in grant applications, which describes efforts to
authenticate antibodies and other key resources required for experiments.
Far-reaching solutions are likely to be hammered out at a meeting hosted by the
Global Biological Standards Institute next September in California, USA.
Want to get info of a specific antibody? Check the web!
In the past decade, various projects have
sprung up to try to make information about antibodies easier to find. The
online reagents portal Antibodypedia (antibodypedia.com), which is maintained by the Human
Protein Atlas, has catalogued more than 1.8 million antibodies and rated the
validation data available for various experimental techniques.
Antibodies-online (antibodies-online.com), another portal, set up a programme two
years ago for independent labs to do validation studies, generally at the
vendors' expense. But out of 275 studies, less than half of the products tested
have made the cut and earned an 'independent validation' badge. The non-profit
Antibody Registry (antibodyregistry.org) assigns unique identifiers to
antibodies and links them to other resources. Another project, pAbmAbs (pabmabs.com/wordpress), operates in a similar way to the
social-recommendation web service Yelp, by encouraging people to review
antibodies.
Antibody validation is now a competitive advantage in the market
The antibody market has grown so much that a reputation for
quality is becoming part of some suppliers’ business plans, which is great news
for us buyers. Several vendors
have announced their own characterization efforts, and some examples are:
1. Abcam is
using a genome editing method called CRISPR–Cas9, which makes precise changes
in DNA. The company is testing antibodies on human cell lines in which target
genes have been disrupted by CRISPR–Cas9 and then posting results for each
reagent tested.
2. Bio-Rad
launched a line of antibodies that have been tested for off-target activity in
western blots against 12 different cell lines.
3. Proteintech
has been using small interfering RNA to knock down gene expression in each new
antibody product — assessing whether the signal subsides with the expression of
the target gene.
4. Abgent
tested all of its antibodies about a year ago. After reviewing the results it
discarded about one-third of its catalogue.
Such efforts are nascent, however, with only a tiny
fraction of companies' catalogues being subjected to validation. Besides, not
all companies disclose the specific conditions of testing, or whether an
antibody has performed poorly under those conditions.
How should I validate my set of antibodies?
For the moment, having an unregulated
market, the validation process relies on researchers itself (although several
companies for this purpose have emerged). Classic techniques, for a ‘in the
lab’ validation, include western blot (WB), immunoprecipitation (IP), siRNA, Immunohistochemistry
(IHC) and Immunofluorescence (IF). Rimm’s lab has even created an algorithm for
antibody validation, which is shown below which can help many people. But the
process is time consuming — Rimm recommends control experiments that involve
engineering cell lines to both express and stop expressing the protein of
interest, for example. Even he acknowledges that few labs will perform all the
steps.
Anyway, antibodies should be evaluated
and the ways and the extent of how this should be done are still open questions
for discussions. Evaluation categories might include:
1.
Knockdown
and knockout approaches to reveal whether an antibody still binds even in the
absence of the target protein.
2.
Tagging
a target protein with a fluorescent marker to reveal whether the antibody also
binds untagged proteins.
3.
Compare a new antibody with a
well-characterized one.
4. Running
the antibody and whatever it binds through a mass spectrometer to analyze bound
molecules for the expected protein fragments.
5. Performing
biophysical analysis for affinity determination and binding kinetics by Microscale Thermophoresis (MST) and/or Surface Plasmon Resonance (SPR).
What other alternatives could we try for validating
antibodies? How can we push companies to do what they should be doing? How can
you help this antibody revolution to go bigger and fruitful?
I leave this questions open and encourage everyone to keep
talking about this so we can make a change, to take good care of your research
by paying attention to the antibodies you buy and use, to share detailed data
on the antibodies you use and how/where it was used in a non-anonymously way
and to put some critical thinking to your experiments and controls.
“The toughest challenge is not so much in antibody characterization but in persuading cell biologists to hold back on using antibodies until these are thoroughly evaluated”
Aled Edwards, University of Toronto, Canada.
1.
Nature 521, 274–276 (21 May 2015) doi:10.1038/521274a
2.
Berglund, L. et al. Mol.
Cell. Proteom. 7, 2019–2027 (2008)
3.
Egelhofer, T. A. et al. Nature
Struct. Mol. Biol. 18, 91–93 (2011)
4.
Nature 483, 531–533 (29 March 2012) doi:10.1038/483531a
5.
Prassas, I. & Diamandis, E.
P. Clin. Chem. Lab. Med. 52, 765–766 (2014)
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