SCIENCE & TECHNOLOGY being tested for many such compounds, he says. "It's what we do not know about the compounds in wastewater that gives me the greatest pause. The few tests for drinking water standards are not adequate when starting with wastewater rather than a pristine water supply" Even reverse osmosis, "the best technology, in my opinion, for making pure water, won't provide a 100% barrier to everything that may be in the water," Brotherton continues. The reason is that seals in the membranes can leak, allowing molecules normally excluded to escape. "What happens in the laboratory does not necessarily replicate itself in normal plant operation," he notes. With regard to the Tampa Bay Area, Brotherton believes the use of reverseosmosis filtration needs to be expanded to ensure high-quality water. "But the local agencies are hesitant to do this, because they don't understand the main problem—the quality of the water people are drinking, not the fact that we're running out of it." At Water Factory 21 in California, ongoing research is directed at fingerprinting bacteria in the highly treated clear-looking wastewater effluent received from the sanitation plant. The idea is to "characterize the range and diversity" ofbacteria in the water that can cause membrane biofouling, explains Harry F. Ridgway director ofwater resources and technology Bacteria are characterized and cataloged according to their 16S ribosomal RNA, a classic, well-established approach. The knowledge gained is expected to be useful in designing membranes that resist biofouling. In Florida, other concerns have surfaced. Switching from groundwater to surface water as the source of potable water may be expected to adversely affect the linings ofwater-distribution pipes, Cental Florida's Taylor indicates. "If you change the water quality, you destroy the film and get the release of undesirable substances." Taylor currently is collaborating withTampa Bay Water on ways to pretreat surface water so this situation doesn't arise. The ultimate goal is to get the last bit of economic efficiency out of all water-treatment technologies, NIST's Pellegrino asserts. "Water is a commodity, and it has to stay a commodity—it's like gasoline in this country now. And there's a finite amount ofwater in the world. When we increase industrial activity and we increase populations, we have to turn water over at a faster rate than nature allows."Treatment processes that run faster and more efficiently and create high-quality water are needed, he insists. • 38
C&EN
/ APRIL
9.
2001
DEATH ROW Calf that is scheduled to be slaughtered because of possible BSE contamination waits in a pen in Freital, Germany.
DETECTING PRIONS Researchers look to improve diagnostic tests used on live animals and patients
T
HE SCOURGE OF MAD cow Dis-
ease, which began in England in the late 1980s and devastated British farmers for most of the 1990s, has found its way to many other European countries. To minimize the spread of the disease, thousands of cattle merely suspected of having been exposed to it have been killed—for lack of a convenient and accurate diagnostic test that could determine for sure whether or not they were infected. Even more ominous is the corresponding lack of a practical and unequivocal diagnostic test for new-variant Creutzfeldt-Jakob disease (vCJD), the human counterpart of mad cow disease. Researchers have been trying for some time to find a rapid, accurate bioassay that would diagnose mad cow disease (bovine spongiform encephalopathy, or BSE), scrapie, vCJD, and other transmissible spongiform encephalopathies (TSEs) in potentially infected animals and people, ideally before symptoms even appear. But progress has been frustratingly slow. TSEs are neurodegenerative diseases that destroy the brain and inevitably cause death. A number of diagnostic tests for these diseases are available. But for the most part they're postmortem procedures, using brain samples obtained in autopsies. The classical method ofTSE diagnosis
is the postmortem histopathologic examination of brain tissue sections by light microscopy. A characteristic spongy appearance of the tissue indicates that TSE-induced degenerative changes have occurred, yielding a positive diagnosis. More rarely, TSEs are diagnosed postmortem by using electron microscopy to look for characteristic "scrapie-associated fibrils" in brain tissue. THE GOLD STANDARD of classical TSE bioassays is a test for infectivity in which brain tissue from a potentially infected animal is introduced into a healthy animal. One then waits to see if the healthy animal gets sick. However, it's slow "If we do this with hamsters it maybe a 'short' 60 days," explained biochemist Bruno Oesch, cofounder of Prionics AG, Zurich, Switzerland. "However, with cattle it can take three tofiveyears. This is not exactly a good way of controlling the disease, especially if you want to prevent BSE-contaminated meat from getting into the food chain." Oesch was speaking at "It's a Mad, Mad, Mad, Mad Cow: Detection and Quantification of Prions," a symposium at last month's Pittsburgh Conference, in New Orleans. The session was arranged and chaired by senior analytical chemist Brian K. Nunnally of EH Lilly & Co. HTTP://PUBS.AC5.ORG/CEN
Anewgeneration ofTSE tests developed by Oesch and others is currently being used to supplement classicalTSE diagnostic procedures. Four such tests were evaluated by European Union authorities in 1999, and three have been approved for use in EU countries. The EU ruled last year that one of the three postmortem tests would have to be used on all cows showing BSE symptoms, and—beginning later this year—on slaughtered cows over 30 months of age. All three approved tests use antibodies to detect prions, the infectious proteins that are the proposed cause of mad cow disease, vCJD, and other TSEs. The antibodies identify prion protein (PrP) by binding to it selectively However, they bind about equally well to both PrP^ (prion protein scrapie), the infectious and protease-resistant form ofPrP, and PrP 0 , the normal, protease-sensitive form of PrP So before the tests are run any PrP Sc in a sample must first be separated from the PrP c inevitably present. The separation is carried out by treating brain tissue samples with protease enzymes. PrP Sc is largely stable in the presence of the enzymes, whereas P r P c is completely destroyed by them. T h e three antibody-based tests approved for use in the EU are: • The Platelia test, an ELISA (enzymelinked immunosorbent assay) procedure developed collaboratively by Bio-Rad (Hercules, Calif) and the French Atomic Energy Commission (CEA), in Fontenay-aux-Roses. • A chemiluminescence ELISA test developed by Enfer Scientific (Kildare, Ireland) and marketed by Abbott Laboratories (Abbott Park, 111.). • Prionics-Check, an immunoblotting bioassay developed by Oesch and coworkers at Prionics. The EU is currently evaluatingfiveadditional tests for postmortem BSE diagnosis. These were discovered by research groups at ID-Lelystad (Lelystad, the Netherlands); Imperial College, London; the University of California, San Francisco (UCSF); PerkinElmer Life Sciences (Cambridge, England); and, once again, Prionics AG. The ID-Lelystad test involves denaturation of samples to increase antibody binding to a key epitope ( binding site). The test can detect TSE in sheep tonsil tissue, and it is currently being tested for detection of human vCJD. The Imperial College test is being developed commercially by Igen (Gaithersburg, Md.) and London-based D-Gen. The test is based in part on specific antibody reagents developed by Imperial College neurogenetics professor John Collinge. HTTP://PUBS.ACS.ORG/CEN
Weiss told the Pittcon session that his The UCSF test is based on a strategy group's studies of the laminin receptor—a for measuring PrPSc that was developed at cell-surface protein that allows prions to UCSF by associate adjunct professor of enter cells—reveals that it is upregulated neurology Jiri Safar, neurology professor (expressed in higher amounts) in BSE catStanley B. Prusiner, and coworkers. "This tle, suggesting that it might be a good approach depends upon looking at an epimarker for disease detection in live anitope that is exposed in native PrPC but mals and potential vCJD patients. He and buried in native PrPSc," Prusiner explained his coworkers are currently at the Pittcon symposium. studying upregulation of the The PerkinElmer test uses diflaminin receptor in blood and ferential extraction and the comcerebrospinal fluid. pany's proprietary Delfia fluoroAnd developmental biologist metric detection technology to Michael Clinton and coworkers test for BSE. And the Prionics at Roslin Institute, in the heart procedure is an ELISA version of Midlothian, Scotland, have of the immunoblotting-based found evidence for a dramatic Prionics-Check test that the EU lowering of a protein called eryapproved earlier. PRUSINER throid differentiation-related A number of other assays are factor in the blood and bone marrow of still in the research stage. For example, TSE-infected animals. They are currently neuropathology professor Adriano Aguzzi trying to find out if this effect also occurs of the University Hospital of Zurich and in human TSEs. coworkers discovered that the The two keyTSE-related pubblood protein plasminogen binds lic health issues are "how can we to PrP S c but not to PrP c —an be sure that the food we eat is interaction that could provide safe and is the human population the basis for a diagnostic test that harboring a reservoir of asympwould Snot require preseparation ofPrP c fromPrP c . tomatic individuals," Clinton tells C&EN. "I would contend that And at the Pittcon sympoto deal with these issues we need sium, prion researcher Stefan a simple blood test effective in Weiss of the University of WEISS the early stages of disease." Munich described the discovery Finally, a promising competitive antiof RNAs that are likewise specific for body-binding assay that uses a novel PrP Sc . Weiss and Corinne I. Lasmézas of extraction method and capillary elecCEA identified a series of RNAaptamers trophoresis to test blood samples is cur(functional molecules) that bind specifirently being developed by research cally to PrP Sc but not to PrP c , suggesting chemist Mary J o Schmerr at the U.S. potential diagnostic applications. One of Department of Agriculture's National the aptamers also interferes with PrP Sc forAnimal Disease Center in Ames, Iowa. mation in cells, indicating that it could have The extraction method, developed by therapeutic uses as well. Schmerr and Andrew Alpert, president of PolyLC, Columbia, Md., is based in part SURROGATE MARKERS, compounds in on a PolyLC technique called hydrophilic blood or other tissues whose presence or interaction chromatography. absence points to prion infection, also represent potential diagnostic targets. For Schmerr notes that the blood of TSE example, a protein in cerebrospinal fluid animals contains PrP Sc , but only in very was recently found to be an indicator of minute amounts. "Traditional test systems brain cell damage. Unfortunately, "at the simply do not have enough sensitivity to moment nobody has been able to prove detect PrP Sc in blood at the concentrations that surrogate markers are highly specific," that are there," she says. "We concentrate Oesch said. "But maybe in the future a our samples about 5,000-fold, and that combination of markers might prove to be brings you into the testing range of capilsomething useful." lary electrophoresis." Her assay requires Oesch noted that "in vCJD the lymonly a 10- to 12-mL sample. phatic system is particularly involved," so "New approaches and detection systems lymphatic tissues are of potential diagwill be required to provide an urgently nostic interest. If a surrogate marker for needed test system for an easily accessible TSEs can be found there, it might permit fluid such as blood," Schmerr says. "I believe diagnostic tests to be carried out on live that good analytical chemistry methodolanimals and patients, since lymph tissue is ogy will produce a practical preclinical test accessible by biopsy for these diseases."—STU B0RMAN C&EN
/ APRIL
9, 2 0 0 1
39
