bio sphere
Detecting prions in blood C
WILLIAM HADLOW, ROCKY MOUNTAIN LABS (RET.)
the number of aggregates, so we have on western blots for 16 of 18 sick hamlaudio Soto and colleagues at the sters but none of 12 healthy hamsters. University of Texas Medical Branch many more free ends for catalyzing the process?” And so the researchers invent- Thus, the assay’s sensitivity was 89%, and the Universidad Autónoma de Maed protein misfolding cyclic amplificaand its specificity was 100%. The assay drid (Spain) have developed the first took several days but was automatbiochemical method for deed throughout. “I think it is very tecting prions in blood (Nat. important that the sonication step Med. 2005, 11, 982–985). can now be automated,” SupatThe work, performed with tapone says. “This makes PMCA hamsters, could pave the way convenient enough to be used by for a blood test for cattle and diagnostic labs.” humans, the researchers say. Shu Chen of Case Western Re“This is easily the most serve University (CWRU) cautions significant advance ever made that the jump from hamsters to hutowards the noninvasive diagmans might not be easy, because nosis of prion diseases,” says details of the amplification mechaSurachai Supattapone of nism—and therefore the assay’s Dartmouth Medical School. exact requirements—are unknown. “The highly innovative tech“Also, in most cases of naturally ocnique is orders of magnitude more sensitive than any other The first biochemical prion detection method for blood is devel- curring human prion disease, the understanding is that infectivity, esrapid diagnostic method.” oped for scrapie. pecially in blood, may be much less Aggregates of prions are thought to cause Creutzfeldt–Jakob distion or PMCA (Nature 2001, 411, 810– than in hamsters,” Chen says. Pierluigi Gambetti of CWRU had ease in humans, “mad cow” disease in 813), which they have now automated some reservations about the work becattle, and scrapie in sheep and goats. and used on blood samples. cause the hamsters were inoculated with Prions are found in the blood of infected In the approach, the researchers ina needle intracerebrally, which could animals and people but at levels that are cubate white blood cells from PrPSc-in10,000–100,000 too low to be roufected hamsters with homogenized brain have introduced PrPSc directly into the tinely detected by current technologies, tissue from healthy hamsters, which pro- bloodstream. He adds that the resuch as western blotting. Soto’s group vides normal prion protein and other searchers should have spiked the hamtherefore set out to amplify the abnorfactors needed for fibril growth. After ster blood with PrPSc to determine the mal protein to detectable levels. “We at30 min at 37 °C, the mixture is sonicatlower limit of detection. Soto says that tempted to mimic what happens in the ed to break up any aggregates of PrPSc. he has addressed the first concern by disease but in an accelerated way so we detecting prions in blood from hamsters Incubation followed by sonication condon’t have to wait months or years,” stitutes one cycle, which is repeated many that were inoculated intraperitoneally. Soto says. And he has now performed the bloodtimes. “We make aggregates grow to a Abnormal prion protein (PrPSc) is a spiking experiment, though the results certain extent, and then come in with a have not yet been published. misfolded version of a normal brain pro- mechanical force that cuts the big pieces Gambetti says that, like any breakinto smaller pieces,” Soto says. As in tein. When PrPSc contacts the normal through, the new protocol must be veriPCR, the product yield increases expoprotein, the latter misfolds. Like beads fied in other laboratories. “But it has nentially with time. on a necklace, PrPSc monomers form the potential to open a new way to deBy using 864 cycles and periodically fibrils, which grow very slowly at their tect extremely small amounts of infectivfree ends. Soto thought, “Why don’t we diluting the mixture into fresh brain ity in bodily fluids,” he adds. a homogenate, the researchers produced mix a step in which we make this unit —Linda Sage enough PrPSc to obtain a positive result grow with a step in which we multiply 14
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© 2006 AMERICAN CHEMICAL SOCIETY
