Nav: Home

Johns Hopkins researchers find widely 'inconsistent' use of antibodies in lab experiments

May 15, 2019

Scientists at the Johns Hopkins Kimmel Center say they have affirmed widespread inconsistencies in the use of a common laboratory procedure called immunohistochemical staining, and say the variations are making many laboratory experiments unreliable.

Their findings were outlined in a special issue of the Asian Journal of Urology.

In a review of papers published about the process and describing the use of antibodies for diagnostic and research applications in biomedical sciences, the investigators set out to document variations in the way scientists practice immunohistochemical (IHC staining).

"Overall, in our experience as journal editors and manuscript reviewers carefully reviewing at least 1,000 manuscripts we estimate, at a minimum, half of them contained potentially incorrect IHC staining results due to lack of best practice antibody validation," says Angelo De Marzo, M.D., Ph.D., professor of pathology, urology and oncology and associate director of cancer research pathology for the Johns Hopkins Kimmel Cancer Center. "It's a problem well known among the scientific community, but many journal editors are not checking this step before publishing affected papers," De Marzo adds.

According to Karen Sfanos, M.S., Ph.D., first author of the study report and associate professor of pathology, urology and oncology at the Johns Hopkins University School of Medicine, IHC is extensively used in research laboratories throughout the world and in clinical pathology laboratories for patient care decision-making. They found that variations were most commonly caused by poor quality and/or lack of proper validation of quality of some antibodies provided by vendors prior to commercialization, and by human error. De Marzo and colleagues are calling for the adoption of industrywide standards for IHC practice that focuses on validation, especially when it comes to human tissue research. Antibodies are blood proteins produced in response to other proteins, called antigens. They are markers for a host of diseases, immune system activities, and reactions to treatments in people and in animal and test tube studies.

At the root of the problem, the researchers say, is a misunderstanding by many investigators and pathologists about the two general classes of antibodies used in IHC staining--clinical grade and research grade. Clinical grade antibodies are highly validated for consistency and reproducibility prior to their use in clinical settings in hospital-based pathology labs, but are limited in number (approximately 500 in use). On the other hand, the great majority of commercially available research grade antibodies used in research laboratories are not held to the same standards of validation as clinical reagents, and the number sold commercially has grown exponentially, with more than 3.8 million different ones available, according to the researchers.

"Results from inaccurate IHC tests published in the scientific literature lead to biased results and call into question the validity of those tests in many research laboratories. Much of our research depends on this even if is not clinical research. Although, it won't kill a person if mouse experiment doesn't turn out okay, there is still a cost. It has been estimated that over $2 billion per year is spent on research antibodies and a significant fraction of this amount, as well as researcher time, gets wasted on unreliable results," says De Marzo. "The problem is so striking it is considered an important aspect of a larger overall problem in biomedical sciences, covered extensively in the press recently, and dubbed the "reproducibility crisis."

A cornerstone of scientific research is the ability of investigators to repeat and reproduce findings of colleagues' studies to either affirm or reject them. If there is unknown variation in test materials, it is virtually impossible to know whether scientists are comparing similar results or not.

While there has been a clear increase in vigilance by some commercial vendors regarding antibody validation, Sfanos said, the rapid increase in growth of commercial antibody companies and offerings makes oversight challenging.

The National Institutes of Health and some scientific journals are beginning to require researchers to provide details regarding what antibodies are used by investigators and what standards were used to verify the consistency and purity of these reagents used in their analyses.

"Some journal editors do not appear to realize the depth and extent of the problem. They don't recognize there is a disconnect between the clinical grade antibodies and the research grade and that all commercial antibodies, even clinical grade, need to be validated in the end user's laboratory before applying it to patient or animal samples in research studies," says De Marzo.

In their review, the authors provide examples of antibody validations they performed, and they direct readers to a growing wealth of literature and online resources, including a novel antibody validation initiative and scoring system being developed, that can help investigators seeking to validate IHC antibodies and assays.
-end-
The 2019 special issue of the Asian Journal of Urology is dedicated to the late Donald S. Coffey, Ph.D., distinguished Johns Hopkins professor and an internationally recognized pioneer in prostate cancer research.

Other authors and contributors included Srinivasan Yegnasubramanian, William Nelson, Tamara Lotan, Ibrahim Kulac, Jessica Hicks, Qizhi Zheng, Charles Bieberich and Michael Haffner.

This work was supported by the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Cancer Center Support Grant (NIH/NCI grant P30 CA006973), the NIH/NCI SPORE in prostate Cancer P50 CA058236, the NIH/NCI U01 CA196390, The Patrick C. Walsh Prostate Cancer Research Fund, U.S. Department of Defense Prostate Cancer Research Program (W81XWH-18-2-0013, W81XWH-18-2-0015; Prostate Cancer Biorepository Network PCBN) and the Prostate Cancer Foundation.

Johns Hopkins Medicine

Related Antibodies Articles:

Antibodies could provide new treatment for OCD
Mental health conditions such as obsessive compulsive disorder could be treated in a new way using drugs that target the immune system, research suggests.
Antibodies in the brain trigger epilepsy
Certain forms of epilepsy are accompanied by inflammation of important brain regions.
Fatal overproduction of antibodies
Bone marrow plasma cells produce antibodies. These comprise two long and two short protein chains.
Antibodies: the body's own antidepressants
Antibodies can be a blessing or a curse to the brain -- it all depends on their concentration.
Antibodies gather and form a circle for defensive attack
Antibodies play a crucial role in our immune system by linking antigen recognition with complement activation for attacking foreign cells.
Hiring antibodies as nanotechnology builders
Researchers at the University of Rome Tor Vergata recruit antibodies as molecular builders to assemble nanoscale structures made of synthetic DNA.
Search for the source of antibodies would help treat allergies
Researchers of Sechenov University together with their colleagues from Russia and Austria summarised everything known about cells producing group E antibodies.
Improving research with more effective antibodies
A new study points to the need for better antibody validation, and outlines a process that other labs can use to make sure the antibodies they work with function properly.
How to enable light to switch on and off therapeutic antibodies
IBS researchers have developed a new biological tool that activates antibody fragments via a blue light.
Ebola antibodies at work
Scientists in Israel and Germany show, on the molecular level, how an experimental vaccine offers long-term protection against the disease.
More Antibodies News and Antibodies Current Events

Trending Science News

Current Coronavirus (COVID-19) News

Top Science Podcasts

We have hand picked the top science podcasts of 2020.
Now Playing: TED Radio Hour

Climate Mindset
In the past few months, human beings have come together to fight a global threat. This hour, TED speakers explore how our response can be the catalyst to fight another global crisis: climate change. Guests include political strategist Tom Rivett-Carnac, diplomat Christiana Figueres, climate justice activist Xiye Bastida, and writer, illustrator, and artist Oliver Jeffers.
Now Playing: Science for the People

#562 Superbug to Bedside
By now we're all good and scared about antibiotic resistance, one of the many things coming to get us all. But there's good news, sort of. News antibiotics are coming out! How do they get tested? What does that kind of a trial look like and how does it happen? Host Bethany Brookeshire talks with Matt McCarthy, author of "Superbugs: The Race to Stop an Epidemic", about the ins and outs of testing a new antibiotic in the hospital.
Now Playing: Radiolab

Speedy Beet
There are few musical moments more well-worn than the first four notes of Beethoven's Fifth Symphony. But in this short, we find out that Beethoven might have made a last-ditch effort to keep his music from ever feeling familiar, to keep pushing his listeners to a kind of psychological limit. Big thanks to our Brooklyn Philharmonic musicians: Deborah Buck and Suzy Perelman on violin, Arash Amini on cello, and Ah Ling Neu on viola. And check out The First Four Notes, Matthew Guerrieri's book on Beethoven's Fifth. Support Radiolab today at Radiolab.org/donate.