October 20, 2002

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 and lymphatic tissue 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 so-called "bioassay," which has a time lag that makes it
impractical for use in detecting prions 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. The 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. Prusiner1., 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 Alzheimer¹s 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 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 antibody¹s
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, 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.
 

The University of California, San Francisco
 
 

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