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[C&EN, Apr. 9, 2001]
Detecting Prions
[C&EN, Apr. 9, 2001]
Detecting Prion Strains
[C&EN, Oct. 5, 1998]
Prion Protein's Role Revealed
[C&EN, Sep. 18, 2000]
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Serono Pharmaceutical Research Institute

Department of Agriculture's National Animal Disease Center

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Claudio Soto

Mary Jo Schmerr

Peter T. Lansbury

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June 18, 2001
Volume 79, Number 25
CENEAR 79 25 pp. 9
ISSN 0009-2347
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Amplification technique improves prospects for diagnostic tests


Mad cow disease, creutzfeldt-Jakob disease in people, and other TSEs (transmissible spongiform encephalopathies) can currently only be diagnosed by postmortem tests on brain tissue. Prions, the errant infectious proteins that cause these diseases, are plentiful in brain tissue but are so scarce in blood and other accessible tissues that detecting them routinely in live subjects has not been feasible.

prion amplificaÉ-2-high-res
PRION AMPLIFIERS Swiss researchers (left to right) Saborio, Soto, and Permanne, have developed a technique (below) that could make TSE diagnoses easier.
But in a study that could make such diagnoses possible, researchers have developed a remarkably simple technique for converting the normal form of prion protein found in the body (prion protein cellular, PrPC) into an abnormal form closely resembling infectious prions (prion protein scrapie, or PrPSc). The technique, called protein-misfolding cyclic amplification (PMCA), is like a polymerase chain reation (PCR) for prions. It was developed by Claudio Soto, head of neurodegenerative diseases research at Serono Pharmaceutical Research Institute, Geneva, and coworkers Gabriela P. Saborio and Bruno Permanne [
Nature, 411, 810 (2001)].

It's "an exciting advance," says research chemist Mary Jo Schmerr of the Department of Agriculture's National Animal Disease Center, Ames, Iowa, who is developing an antibody-binding assay to test for prions in blood. PMCA could "make it possible for some of the present technologies to be used for testing for TSE infections prior to clinical disease [and] for looking at cross-species transmissions of these diseases. And because of the short time needed to do the amplification, the answers from these experiments may be obtained in days, rather than in years."

Prions replicate in the body by somehow inducing normal prion protein to change its conformation to the abnormal form, similar to the way a cult might recruit adherents. PMCA was developed in an effort to mimic this process in vitro.

In PMCA, brain tissue containing normal prion protein from an uninfected animal is mixed with tissue containing an aggregated form of infectious prions. The normal form is converted to the abnormal form, making the aggregates grow larger. The aggregates are then sonicated to increase their surface area, enhancing their ability to mediate, or "seed," further prion formation. Rounds of conversion and sonication are repeated several times.

In the paper, a 58-fold average amplification was achieved, but the Serono group has since boosted that to the several-hundredfold range. And the researchers have obtained preliminary data indicating that PMCA can detect trace levels of prions in blood.

The first technique for converting normal to abnormal prion protein in a cell-free system was devised several years ago by associate professor of neurology Peter T. Lansbury, of Harvard Medical School; Byron Caughey of the National Institutes of Health's Rocky Mountain Laboratories, Hamilton, Mont.; and coworkers [Nature, 370, 471 (1994)]. However, the degree of conversion was very low, and the researchers weren't able to detect any new prion infectivity caused by the conversion.

A key reason why PMCA's conversion efficiency is higher is that it uses brain homogenate as PrPC starting material, whereas highly purified, recombinant PrPC was used as a starting material in the Lansbury-Caughey technique. "We have strong evidence," Soto says, "that there is a factor in the brain homogenate that catalyzes the conversion."

Another major difference is that in PMCA "we use cycles of incubation and sonication, letting the aggregates grow and then breaking them down to multiply the number of converting units," Soto says, "whereas the earlier conversion technique was a one-step process."

Serono intends to license PMCA to other companies for use in prion diagnostic tests of blood, brain, and other tissues.


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