NEW YORK(GenomeWeb) – Antibody provider Abcam said last week that it has begun knockout validation of its reagents.
Coming from a prominent vendor, the move marks a step forward in the growing effort to improve antibody quality as the life science community becomes increasingly aware of the significant research time and money being wasted due to faulty affinity reagents.
"We felt that the industry needed a response to [questions about antibody] specificity," Abcam CEO Alan Hirzel told GenomeWeb. And, he noted, the emergence in recent years of CRISPR gene-editing technology has made knockout validation — in which antibodies are tested for specificity in cell lines in which their target protein has been removed — feasible on a large scale.
To enable its new validation effort, Abcam has signed an exclusive agreement with Horizon Discovery Group, which will provide the company with the haploid knockout cell lines it needs. Obtaining haploid, as opposed to diploid, knockout lines was a key point, Hirzel said, in that they provide "a cleaner on-off result with no compensation."
Abcam signed the exclusive deal with Horizon this summer and currently offers 23 antibodies that have gone through knockout validation.
"What we are focused on now is getting a good understanding of how the technology works and being able to explain that to researchers and then rapidly scaling up how many of the antibodies we have validated in this way," Hirzel said, adding that he hopes to offer on the order of several thousand knockout-validated antibodies in the next few years.
Under the agreement, Horizon provides Abcam with knockout cell lines and "we do all the validation ourselves using a combination of fluorescent western blots and other fluorescent techniques to make it visually obvious to researchers that this is a true negative control," Hirzel said.
The deal allows Abcam to maintain its exclusive access to Horizon's technology for antibody validation for as long as Abcam chooses to renew the license, he added.
Hirzel said that while "it is still early days" initial researcher response to the offering has been "extremely positive."
In a recent interview with GenomeWeb discussing issues of antibody quality, University of Toronto researcher Aled Edwards cited knockout validation as a key step in establishing an antibody's validity, but one which, due to the challenges involved, was not commonly done.
Edwards and his colleagues recently put forth a method using mass spec to validate antibodies for immunoprecipitation experiments, but, he noted, that while that approach was effective for IP work, knockout experiments were required to confidently validate antibodies for methods like immunofluorescence.
Told this week of Abcam's decision to begin doing knockout validation, he said it was "definitely the right direction" for vendors to take.
Reagents vendor Proteintech began offering knockdown validation of its antibodies last year. As opposed to knockout, in which the gene for the target protein is edited out of the cell's genome, knockdown validation uses methods (siRNA in Proteintech's case) to silence the gene producing the target protein.
According to Proteintech CEO Jason Li, the company currently offers 800 antibodies validated in this way.
Hirzel said that in discussions with researchers, Abcam found that many preferred knockout validation as opposed to knockdown due to the fact that the latter approach can still leave a faint signal.
However, he added that "anything the industry is doing to provide better validation that these products work the way they should, should be welcomed."
Hirzel suggested that the growing concern about antibody quality stems in part from an improved ability to assess this quality.
"The expectation of good performance is ever increasing as tools and techniques for determining if these antibodies are working the way they are supposed to improves over time," he said, noting that, in the early days of antibody research, polyclonal antibodies were often referred to as "probability reagents."
"It was almost acknowledging that there was a very good chance the antibody was binding to what it was supposed to, but you were never quite certain," he said. "I think as these technologies like knockout come along it just takes some of the uncertainty out of the story."
Proteintech's Li likewise painted his company's increased validation efforts as a matter of technology development.
"The technology has developed to a certain degree that allows this to happen, and when we saw this we decided to go for it," he said. "With siRNA there was a lot of trial and error for a long time. Now we have been able to master that and can get a lot of positives instead of trial and error. So it is basically in-house technology development."
Neither Abcam nor Proteintech are charging more for antibodies featuring these new levels of validation, the thinking in Li's case being that "because it is the responsibility of the manufacturer to get the product right, therefore you should do it if the technology is available."
He added that he believed such approaches would become standard for antibody vendors in the future.
Antibody quality is a pressing issue for the proteomics field and for life sciences research in general. In a recent commentary in Nature, researchers Andrew Bradbury and Andreas Plückthun, of, respectively, Los Alamos National Laboratory and theUniversity ofZurich, noted that a 2008 study found that fewer than half of the roughly 6,000 commercial antibodies commonly used were specific to their stated targets. They estimated that $800 million is wasted annually on faulty antibodies.
"The current standards of the market are not right," Li said, noting that end users too often assume a reagent will work as expected, while distributors and resellers rely on the original manufacturer to ensure their reagents are of high quality. This, he said, opens the door for some manufacturers to pass off bad antibodies more or less anonymously by selling them under the name of a reseller.
A notable example of this phenomenon is the case of USCN Life Sciences, a Wuhan, China-based antibody firm that several researchers, including Nader Rifai, director of clinical chemistry at Children's Hospital Boston and editor-in-chief of Clinical Chemistry, have identified as a purveyor of faulty antibodies.
Despite a number of red flags, including, Rifai said, a lack of meaningful reference material accompanying its antibodies and the large number of antibodies, including quite obscure proteins, offered, USCN has managed to get distribution of its products from a variety of reputable resellers, and these products have turned up in the scientific literature.
For instance, in 2012 researchers from Harvard Medical School and the University of Alabama published a letter to the editor in the American Journal of Nephrology in which they expressed concern about a previously published study using a USCN ELISA for measuring HJV protein.
In their letter, the researchers wrote that through efforts to replicate this study, they had determined that the "USCN HJV ELISA does not detect human or mouse HJV, but is instead recognizing some other protein(s)."
The case of USCN suggests that, while validation work like those initiated by Proteintech and Abcam are welcome, the extent to which their efforts can protect researchers from bad antibodies is limited.
As Rifai noted in a 2013 interview discussing USCN, when going outside reputable vendors for supplies, researchers would do well to do their own validation.
"I wish fraudulent companies did not exist, but if scientists do what they are supposed to do, they can save themselves a lot of heartache," he said.