Nature literature reveals big problems with antibodies - Database & Sql Blog Articles

Many biomedical experiments are performed on antibodies. Therefore, the upper limit of many experiments depends on the quality of the antibody. Rotten antibodies can lead to erroneous results and conclusions, and even eventually destroy a project.

Everything was very pleasant to the pathologist David Rimm of Yale University in 2006. He developed a biopsy test method for evaluating the treatment of skin melanoma. This method relies on the binding of a large Y-type antibody to a particular biomolecule to label its structure on the sample. Rimm found that the antibody binds to the biopsy tissue of the tumor, producing a specific structure that can be used to assess whether the patient needs to undergo strict medical treatment after surgery. He invested $2 million in the establishment and advancement of the test method.

However, in 2009, the situation turned sharply. Rimm ordered a new batch of antibodies, but his team had difficulty repeating the results. Antibodies are purchased from the same company, with the same item number, different batches, but they cannot be localized to the specific structure at the same location, or even stained on the same tumor. Rimm was forced to abandon his project development on melanoma antibody testing. He said: "This is a lesson. We should not be so dependent on antibodies. This is a frustrating meeting."
Antibodies are a common tool in the biological sciences and can be used to identify and isolate other molecules. However, antibodies are also considered to be a common cause of problems in research. This batch-to-batch variation of this antibody experienced by Rimm leads to a difference in dramatic results. More seriously, antibodies often recognize proteins other than the protein of interest, which can lead to project failures, time, money, and large waste of samples.
Many people believe that antibodies are the main reason for the "repetitive crisis" in the current experiment. People gradually realize that the results of biomedical experiments are difficult to repeat, and the conclusions based on these experiments need to be further confirmed. In his controversial, analytic article, Glenn Begley, chief scientist at TetraLogic Pharmaceuticals in Pennsylvania, pointed out that antibodies with poor specificity are more prone to problems than other tools in the experiment, and 47 of the 53 cancer research papers. The results are difficult to repeat.
Nowadays, scientists who have a small amount of antibodies due to antibodies have raised this problem. Rimm, who was frustrated by the antibody problem, began his "jihad", wrote relevant reviews, held webinars, and raised this issue in countless conference discussions. These scientists have called for a set of standards for the production, use, and description of traits in antibodies. At the same time, there are many grassroots research efforts to measure the quality of antibodies, which have sprung up.
But the time is too short to form a real reform. "Although there are so many resources, no one uses them, and many people don't even know them," said Len Freedman, head of the Global Biological Standard Institute, a Washington-based non-profit organization. "Many antibodies Suppliers don't want to change the status quo, even if some antibodies are very poor." Buyers pay attention to Loannis Prassas, a proteomics researcher at Mount Sinai Hospital in Canada, and his colleagues working on a protein called CUZD1, which they believe can be used to characterize pancreatic cancer. Whether it is sick or not. They bought a protein test kit, which took two years, $500,000, and thousands of patient samples found that the antibody recognized another tumor protein, CA125, rather than identifying CUZD1. Prassas recalls that he and his team were eager to prove a promising hypothesis that led to no experiments on antibody validation. If someone says "You can use this test", you will be eager to do the test and forget that it is not your situation.
Most scientists believe the text on the label of the antibody tube, Rimm joked: "As a pathologist, I was not trained to validate these antibodies, only knowing to order them."
Antibodies are produced by most vertebrate immune systems against invaders such as bacteria. Since 1970, scientists have developed a large number of antibodies for research. The researchers injected the protein of interest into the rabbit, and the B cells in the white blood cells began to produce antibodies against the protein, and the blood of the rabbit was collected to obtain antibodies. In order to obtain a more consistent product, B cells can be recovered and fused with an "immortal" cell (myeloma cells) and cultured in vitro. In theory, a steady supply of antibodies can be obtained. Thirty years ago, scientists needed to extract antibodies for research. From the late 1990s, reagent companies began to take over the business. Today, more than 300 companies are selling more than 2 million antibodies for research. According to a report by the global consultancy Frost & Sullivan, the total antibody market reached $1.6 billion in 2011. Destructive effects There are indications that antibody problems have widespread and potentially devastating effects on research records. In 2009, a journal dedicated to evaluating the quality of antibodies used to study G-protein coupled receptors (GPCRs). The G protein-coupled receptor is a cellular signaling protein that is a target protein for various diseases such as incontinence and schizophrenia drugs. Analysis of 49 commercial antibodies targeting these 19 signaling receptors revealed that most of them could bind multiple proteins, indicating that they cannot be used to specifically recognize receptors of interest.
Epigenetics is a highly antibody-dependent discipline that requires antibodies to recognize how proteins regulate gene expression. In 2011, an assessment reported that 1/4 of the 246 antibodies used in epigenetic studies faced antibody-specific challenges, suggesting that they often bind to other proteins. Four of these antibodies have high specificity but are not at the correct target protein.
Interestingly, scientists know that some antibodies are not credible in their field of research, but it is very difficult to assess the problem in the entire biological field. Perhaps the biggest assessment now is from the Swedish consortium Human Protein Atlas, which works to assemble antibodies to every protein in the human genome. It subsequently found that less than 50% of the nearly 20,000 commercial antibodies were able to bind effectively to tissue sections. This has led some scientists to suggest that more than half of the commercial antibodies are not credible. But credibility is closely related to experimentation. In collaboration with Human Protein Atlas, Mathias Uhlén from the Royal Institute of Technology in Stockholm said: "We found that commercial antibodies are generally available, but in others' experiments, they may not work."
Researchers should check that an antibody has been tested in a particular application and tissue type, but the quality of the product information provided by the supplier varies greatly. Researchers often complain that reagent companies do not provide antibody-specific data or batch-to-batch differential data. The company may provide a batch of antibodies but give you information about the previous batch of antibodies. These data are often derived under ideal conditions and do not reflect typical experimental conditions. Antibody companies explain that it is not possible to test their products under each of the experimental conditions, but the data they provide is credible and will work with researchers to improve antibody quality and performance.
"Many scholars use Google to find products, so for some companies optimizing search results is more important than optimizing products," said Tim Bernard, chief of British biotechnology consulting firm Pivotal Scientific (based at Frost & Sullivan in Washington). "Researchers tend to be more concerned. It’s the 'Amazon effect' that they want to get antibodies, they want to receive the goods in 2-3 days, and they are free of mail. "Researchers who are aware of antibody problems say scientists need to be more vigilant," antibodies are not Magic potions, you can't add them to your samples, and expect the results to be 100% credible without rigorous thinking," making neuroscience-related antibodies, said James Trimmer, chief of NeuroMab at the University of California, Davis. Like many suppliers, NeuroMab clearly identifies the types of experiments that antibodies can be applied to, but researchers do not always follow them. Ideally, researchers will refuse to buy antibodies that do not have extensive validation data, or will verify antibody titers by themselves (see "Bad antibodies"). This is what Rimm is currently working on. He introduces each interested person to a newly developed multi-step process for antibody titer evaluation. However, this process is very time consuming. For example, Rimm recommends setting up a control experiment to establish a cell line that expresses and does not express the protein of interest using cell engineering. He even admitted that almost no lab would perform all the steps of the evaluation process. Some researchers buy half a dozen antibodies from different suppliers and then perform a preliminary experiment to screen for antibodies that are active. But they may just buy the same antibodies from different places. Large reagent suppliers are highly competitive in the catalog, so they buy antibodies from small suppliers and relabel them. Bernard said that 2 million antibodies on the market may have only 250,000-500,000 specific "core" antibodies. For some reason, researchers believe in the literature or published literature. But this led to a self-sustaining problem, and subsequent development of antibodies with better potency was hardly adopted, said Fridtjof Lund-Johansen, a proteomics researcher at the University of Oslo. "We have a good market. Antibodies "," but we don't know which ones are, "Lund-Johansen is developing a high-throughput assay that can test the titers of thousands of antibodies at once. Test time Over the past decade, various projects have sprung up and are dedicated to providing more convenient antibody search information. Antitrustpedia (Antibodypedia.com), an online portal created by Human Protein Atlas, records more than 1.8 million antibodies and provides their test data for different experiments. Another online portal, Antibodies-online (Antibodies-online.com), has launched a project to commission independent laboratories to test antibodies two years ago, most of which are funded by vendors. However, less than half of the 275 studies met the criteria and were recognized as "independent tests." The non-profit Organization Registry (antibodyregistry.org) assigns a unique identifier to the antibody and then directs it to other resources. Another project, pAbmAbs (pabmabs.com/wordpress), operates in a manner similar to the socially recommended service page Yelp, encouraging people to provide comments on antibodies. But these efforts have not yet found a foothold in the scientific community, and many of the scientists mentioned in this article do not even know the existence of these resources. Antibodies are not magic reagents The antibody market is already very crowded, which has led some suppliers to take good quality credit as part of their business plans. Bernard said: "The competition is so fierce now, we must differentiate products." Abcam, Inc., of Cambridge, UK, encourages antibody users to report their own usage data on their company's website. Abcam's analysis of purchase behavior showed that customers viewed an average of nine data pages before purchasing antibodies, indicating that customers need more data. Abgent, an antibody company based in San Diego, Calif., and its subsidiary Shanghai, China, Wuxi WuXi PharmaTech, launched an antibody testing program about a year ago. After review, it cut 1/3 of the antibody product in the catalog. Whether this is a wise decision depends on whether the customer is willing to spend more money to buy a better product, the company's regional leader John Mountzouns said, but at present, the customer complaint rate has dropped significantly. Some scientists are calling for more radical changes. In a February review of Nature, Andrew Bradbury from New Mexico, Los Alamos National Laboratory and more than 100 co-authors presented a disruptive approach to antibody production and marketing. They recommend using only antibodies that have been defined at the DNA sequence level and producing antibodies by tissue engineered recombinant cells. This can greatly reduce the differences in the production of animals from antibody production. But this proposal requires the use of specific antibody information that many companies consider to be trade secrets. This will lead to the dismantling of the antibody market and millions of products.
Uhlén, the co-author of this review article, believes this plan is a distant hope. He believes that Bradbury's expectation of "recombinant antibodies" will require 10-100 times higher than traditional methods, and there is no guarantee that antibody titers will be better than traditional methods. "In the end, antibody binding potency is more important," he said. "There is no clear sequence that does not work." In order to find cheaper, faster, more reliable ways to produce antibodies, there are other projects going on. If the immunized animal is not used, the antibody protein sequence is expressed and optimized only on the virus. It is imperative to describe the situation of antibodies available today. Antibodies are an important component in improving the repeatability of experiments, and researchers have proposed to establish a commercial antibody certification program in an independent institution. At the same time, many journals (such as Nature) require the author to provide the basis for the detection of antibodies used in the article for specific experiments. The quality of antibodies will slowly increase rather than leap. Trimmer said he hopes to form a benign feedback mechanism: when researchers see some illusions, they will further verify the results and discover more illusions. He said that the widespread unrecognized antibody testing is now fading, "things are beginning to change," he said. "We need to discuss this topic all the time."

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