UCSF-led researchers have developed a highly sensitive, automated test for detecting prions (PREE-on) that they report significantly improves the accuracy and speed of detecting the various forms of the infectious agent, which causes a set of neurodegenerative diseases, in cattle, sheep, deer and elk. Because the test is automated, the researchers say, it could be used for high-throughput testing of brain samples of cattle with bovine spongiform encephalopathy (BSE), or "mad cow" disease, as well as deer and elk with chronic wasting disease (CWD).
The test, an immunological probe, or "immunoassay," uses a novel strategy and newly developed, high-affinity antibodies to reveal and measure prions in brain tissue. As a result, it is able to directly measure infectious, abnormal prion protein.
The high sensitivity of the test in detecting BSE and CWD prions, reported in the October 21 on-line version of Nature Biotechnology, culminates an effort to perfect the application of a principal that the UCSF team firsts reported in 1998 in a study in hamsters.
Known as a conformation-dependent immunoassay (CDI), the test is able to detect much smaller levels of the infectious prion protein than can be seen with the current standard immunological procedures. Those older methods, which detect only fragments of infectious prion protein that are resistant to an enzyme known as protease, are currently used in the United Kingdom and Europe to detect prion-infected brain in cattle.
The new test in fact matches the sensitivity of what is currently the most reliable technique for determining the level of prion infectivity in a tissue. This bioassay, which has a time lag that makes it impractical for the rapid detection of prions in large-scale testing in tissue, involves injecting brain tissue from cattle with BSE into mice genetically engineered to over-express bovine prion protein. The expression of the bovine prion protein makes the mice highly sensitive to bovine prions from infected cattle. UCSF researchers previously reported that they had developed a bioassay for infectious prions in genetically engineered (or transgenic) mice. In the current study, the researchers report that this bioassay for infectious prions in genetically engineered (or transgenic) mice may detect up to 10,000-fold more prions than standard bioassay in normal mice.
"The conformation-dependent immunoassay essentially lowers the threshold for detection of bovine spongiform encephalopathies," says the lead author of the study, Jiri Safar, MD, UCSF associate adjunct professor of neurology and a member of the UCSF Institute for Neurodegenerative Diseases, which is directed by co-senior author Stanley B. Prusiner(1), MD, UCSF professor of neurology and biochemistry.
"We believe that by applying the test to cattle we should significantly reduce human exposure to bovine prions," says Safar. In addition, he says, while scientists do not know whether chronic wasting disease in deer and elk can be transmitted to humans, the new test "offers a very important first step toward being able to diagnose chronic wasting disease early and to study the biological properties of CWD prions."
More broadly, says Safar, the CDI could be applied to studies of other neurodegenerative diseases, such as Alzheimer's disease, that also involve the transformation of normally shaped proteins into abnormal forms. The goal of such studies, he said, would be to detect the development of transformed proteins before the symptoms of a neurodegenerative disease develop.
In the current study, the CDI was used to detect infectious prion protein in brain tissue samples taken from BSE-infected U.K. cattle, and U.S. CWD-infected deer and elk. In 1,729 tests, the CDI correctly identified samples of diseased and normal tissue with 100 percent accuracy.
In its current capacity, the CDI test could be used in Great Britain and Europe to detect BSE prions in cattle before potentially contaminated meat enters the human food supply. In the United States, it could also be used to test deer and elk for chronic wasting disease prions.
However, the ultimate goal of the technology, the researchers say, would be to apply the assay to testing for prions while animals are still alive, perhaps using blood or some peripheral tissue such as muscle. Early evidence in hamsters and mice suggest this might be possible. In this case, the test could also potentially be used to diagnose patients with one of the several human forms of prion disease, known as Creutzfeldt-Jakob disease.
The need for a more sensitive test
The need for a more sensitive test to detect infectious prion proteins stems from the fact that studies increasingly show that the bioassays traditionally used to detect whether animal tissue is infected are not sensitive enough, and therefore have likely misrepresented the frequency of animals being infected.
In the current study, the researchers report that while the concentration of BSE prions in brain tissue was 1,000 infectious units per gram when measured in normal mice, it was 10,000,000 infectious units per gram when measured in mice genetically engineered to express multiple copies of the bovine prion protein gene. This finding is of concern because early on in the BSE epidemic in Great Britain decisions on what precautions to take were based on titrations in normal mice. Thus, says Safar, they underestimated the likely threat of infectivity in many organs.
"This finding indicates that previous attempts to quantify BSE and scrapie prions in milk or non-neural tissues, such as muscle, may have underestimated infectious titers by as much as a factor of 10,000, raising the possibility that prions could be present in these products in sufficient quantities to pose risk to humans," says Safar.
The new transgenic mice, developed in the Prusiner lab, provide information about infectivity within 220 to 400 days, thus accelerating the accumulation of data. The Prusiner lab is now using the mouse model to test tissue samples for the UK Department of Environment, Food and Rural Affairs.
"At present, we have no data on the frequency of sub-clinical prion infections in livestock," says Safar. "Because most livestock destined for human consumption are slaughtered by two years of age, many animals may be infected but never show clinical signs of central nervous system dysfunction since incubation periods generally exceed three years."
"The high sensitivity of the CDI, and the availability of a manual or automated version to test large numbers of animals may profoundly alter our view of the epidemiology of prion diseases," says Safar.
The study was done in conjunction with researchers at The Scripps Research Institute, including senior author R. Anthony Williamson PhD, and Dennis R. Burton, PhD, in the Department of Immunology and Molecular Biology, whose team developed the high-affinity antibodies used in the test.
In 2001, UCSF licensed the technology for CDI, developed in the Prusiner lab, to InPro Biotechnology Inc., of South San Francisco, California, which Prusiner founded. Prusiner, Safar and some other members of the Institute for Neurodegenerative Diseases are scientific advisors to, or own stock in, the company.
The automated CDI is one of several immunoassay tests currently being evaluated by the European Community in a formal validation trial. However, it is the only test that is not based on the traditional detection of the protease-resistant fragment of infectious prion protein. The test received a perfect record in the first part of the trial, which involved testing 200 brainstem samples provided by the EC. The results are posted at: http://www.irmm.jrc.be/. A field trial, currently underway, involves testing the test in numerous labs throughout Europe. The UCSF team hopes that the second half of the validation, which is being performed by scientists at InPro Biotechnology, will be completed some time this winter.
How the conformation-dependent immunoassay works
One of the many challenges in attempting to detect infectious prion protein is distinguishing the infectious form from the normal prion protein that exists in a healthy state in humans and animals.
The standard technique, developed in the Prusiner lab 20 years ago, involves using an enzyme known as a protease to destroy normal prion protein (PrPC), which is ubiquitous in brain tissue. Once this occurs, scientists apply fluorescently lit antibodies that react with residues of the relatively resistant abnormal prion protein (PrPSc), thereby highlighting it.
The limitation of this technique is that scientists have since learned that there is a large part of the abnormal prion protein that is protease sensitive, and that portion escapes detection by the standard technique. Thus, this traditional method underestimates the level of PrPSc in tissue.
The new approach involves revealing the region of PrPSc that is exposed in the normal PrPC but is buried in infectious PrPSc, using high affinity, newly generated antibodies that identify PrPSc through the distinct shape of the molecule, independent of proteolytic treatments. This makes it possible to detect potentially large concentrations of protease sensitive PrPSc molecules.
The first step in using the immunoassay involves exposing a tissue extract containing infectious prion protein in its natural state to the antibody and measuring the reactivity. Next, the prion protein is unfolded by chemical means so that the hidden region is exposed. Predictably, the antibody's immunoreactivity to this denatured region, as measured by its degree of binding to the molecule, is much higher than it is to the diseased protein in its native state. The ratio of denatured to native infectious prion protein indicates the amount of PrPSc.
The test offers the potential for rapid prion strain typing, which is an essential tool for identifying those prion isolates that are readily transmitted to humans, says Safar. There is a theoretical possibility that BSE may have infected not only cattle but also sheep, he says. Because BSE is transmissible to humans and scrapie is not, the ability to identify BSE in sheep and to distinguish it from scrapie is increasingly important.
The study is funded by the National Institutes of Health.
Other co-authors were Michael Scott, Jeff Monaghan, Camille Deering, Svetlana Didorenko, Juile Vergara, Haydn Ball, Giuseppe Legname, Hanna Serban and Darlene Groth of the UCSF Neurodegenerative Research Institute; and Estelle Leclerc and Laura Solforosi of The Scripps Research Institute.
(1) Prusiner won the Nobel Prize in Physiology or Medicine in 1997 for discovering that a class of neurodegenerative diseases known as spongiform encephalopathies are caused by prions.
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