BSE Strain Test

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This information was on Tom Pringle's Mad Cow site:

Researchers develop rapid and sensitive prion test

October 1998

Nature Medicine Press Release

A team of researchers led by Nobel Prize winner Stanley Prusiner has

developed a test to distinguish between different strains of prion-the

infectious agent believed to be the cause of BSE in cattle and the

neurodegenerative Creutzfeldt-Jakob's disease in humans. It is important to

differentiate between prion strains not only to better understand their

biological properties, but also because some strains are thought to result

in disease more quickly than others.

The new test represents a major advance because it allows sensitive and

rapid differentiation between prion strains in the test tube, compared with

existing methods that require year-long studies in mice. The fluorescent

immunoassay test works by distinguishing between the unique protein

conformations of the different strains.

In an accompanying News +ACY- Views article, o Aguzzi of the Institute

for

Neuropathology in Switzerland discusses the role of prions in disease and

writes that the +ACI-original and clever+ACI- test will be of +ACI-utmost

interest for

public health.+ACI-

University of California-san Francisco researchers report test that detects

prion diseases, illuminates novel findings about infectious prions

September 28, 1998

University of California, San Francisco Press Release

Researchers at The University of California San Francisco report that they

have developed a highly sensitive, rapid technique for detecting the

infectious agents that cause prion diseases. And they said they expect the

assay will ultimately be useful for detecting prions causing +ACI-mad cow+ACI-

disease and Creutzfeldt-Jakob disease in humans.

With automation, they said, the tool could be applied to commercial testing

of meat, biological and pharmaceutical products.

+ACI-This is an extremely exciting scientific breakthrough,+ACI- said the lead

author of the study, Jiri Safar, MD, an associate adjunct professor of

neurology at UCSF. +ACI-We still have some scientific aspects of the assay to

resolve, but we are moving from a scientific discovery to an engineering

challenge.+ACI-

But the significance of the UCSF study, reported in the October issue of

Nature Medicine, extends beyond the hope for an effective screening tool.

For the assay has revealed stunning insights into the nature of the novel,

inscrutable pathogen that causes +ACI-mad cow+ACI- disease, Creutzfeldt-

Jakob's

disease in humans and a variety of other neurodegenerative diseases seen

across species and known collectively as spongiform encephalopathies. The

findings have given the researchers new direction for exploring the way in

which the pathogen, called prion (PREE-on), for proteinaceous infectious

particle, functions.

The test tube immunossay, which so far has been used to detect infection in

hamsters, identifies extremely low levels of prion proteinthe only known

component of the infectious prionand does so within a matter of eight hours.

And the researchers said they believe the design can be adapted for

large-scale robotic processing.

By contrast, current detection models, called bioassays, involve inserting

suspected infectious tissue into the brains of laboratory animals and

observing them for development of the disease. The process takes between 60

to 180 days, and cannot be conducted on a large, commercial scale.

The new technique, conducted in plastic plates, is also expected to prove

effective for diagnosing new-variant Creutzfeldt-Jakob disease (CJD) in

living patients. Scientists fear that some 25 people in Great Britain and

France may have developed the disease by eating tainted meat in the 1980s.

But the insights the test offers into the biology of the prion protein are

consuming much of the researchers' attention. Previous research has revealed

that all mammals examined contain normal, benign prion protein, and it is

believed that they only become destructive when the prion protein changes

shape, from a coiled structure to a flat sheet. The conversion in the

infectious form of the disease (which can also be inherited or occur

spontaneously) is believed to occur when already infectious prion protein,

or PrPSc, clasps onto the normal prion protein, or PrPC, twisting it down

flat in a morbid, fateful dance.

The researchers developed an assay that detects a region of PrPSc protein

that, while exposed in normal PrPC protein, becomes tucked, or folded, in

the diseased PrPSc molecule. Fluorescently labeled antibody that reacts with

the folded region of PrPSc only after the disease protein is unfolded, or

denatured, is used in the assay.

The researchers first expose a tissue extract containing infectious prion

protein in its natural state to the antibody and measure the reactivity.

They then unfold the prion protein by chemical means so that the hidden

region will be exposed. Predictably, the antibody's immunoreactivity to the

denatured region, as measured by its degree of binding to the molecule, is

much higher than it is to the diseased protein in its natural state. The

ratio of denatured to native infectious prion protein indicates the amount

of PrPSc.

The researchers used the model to test brain tissue taken from hamsters

infected with eight different strains of prions. They plotted the results as

a function of the concentration of PrPSc for each strain. And their findings

were dramatic. Like seemingly insignificant holes cut in paper can create

the image of a snowflake, the points on the graph revealed detail about the

proteins' unique properties that the molecular biologists couldn't see on

their own: specifically, that each of the eight different strains of

infectious prions had unique shapes.

Researchers have known that prion diseases, even within species, vary in

length of incubation, topology of prion accumulation and distribution of

accumulated protein deposits in the brain. But while they have suspected

that these variations, or strains, were represented by different protein

shapes, they have never had direct evidence. Moreover, it has long been

believed that a protein has only a single conformation, as determined by its

amino acid sequence, and all eight strains did represent a single molecular

sequence.

+ACI-We know that PrPC and PrPSc have very distinct shapes. What has become

clear is that while all of the strains contain a common molecular sequence,

each protein strain has a distinct shape,+ACI- said Fred E. Cohen, MD, PhD, a

professor of pharmacology and medicine at UCSF and a co-senior author of the

study.

The assay also revealed that PrPSc protein contains a protease-sensitive

fraction, which surprised the researchers. +ACI-We always thought PrPSc was

strictly protease resistant,+ACI- said Stanley B. Prusiner, MD, a professor of

neurology, biochemistry and biophysics at UCSF, the winner of the 1997 Nobel

Prize in Physiology or Medicine, and the other senior author of the study.

In an effort to tease out the component of prion protein that might actually

confer the most crucial distinction in strainsthe time it takes for the

disease to developthe researchers plotted the protease-sensitive component

of the PrPSc versus incubation time and were struck by what Safar called 'a

gorgeous straight line.'

+ACI-Until now, we believed that once formed in the brain, prions could not be

degraded. We now understand that it is the rate at which prions are degraded

that explains the differences in the time that it takes a prion strain to

cause disease,+ACI- said Cohen. +ACI-Since the body can begin to clear the

proteinaceous mess from the brain, treatments are being developed to assist

this process.+ACI-

+ACI-The only conclusion,+ACI- Cohen said, +ACI-counterintuitive as it is, can

be that

the rate-limiting step in prion replication has little to do with PrPsc.

Instead, Cohen and Prusiner suggested, it must have to do with an earlier

stage in the development of PrPSc, when normal PrPC protein binds to an

as-yet-elusive +ACI-protein X.+ACI- Protein X is believed to act as a

molecular

chaperone, moving the normal protein out to the dance floor where it

presumably is handed off to its deadly suitor.

Needless to say, the researchers are turning their attention to this earlier

stage in the conversion cascade, before the protease-resistant fraction is

formed.

+ACI-While we still can't visualize protein X, we need to see if we can figure

out its role,+ACI- said Safar. The researchers' challenge, which molecular

biologist face every day in their explorations, will be developing still

more clever techniques that will reveal to them what they can't actually

see, in this case the machinations of a deadly protein.

The University of California has filed a patent on the full technology

platform for the immunoassay. Centeon Inc.. holds a license granting them

exclusive rights to the immunoassay technology.

Other co-authors of the UCSF study included Holger Wille, PhD, Vincenza

Itri, BS, Darlene Groth, BS, Hana Serban, MS, and Marilyn Torchia, DVM. The

study was supported by grants from the National Institutes of Health, as

well as well as by gifts from the Leila G. and Harold Mathers Foundation,

Sherman Fairchild Foundation and Centeon.