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New Prion Research

Mike

Well-known member
Detection of Prion Aggregates
Posted December 11, 2006
Man-Sun Sy, Ph.D., Case Western Reserve University School of Medicine, Cleveland, Ohio

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are caused by the conversion of the endogenously occurring prion protein (PrPC) to the disease-causing prion protein conformation (PrPSc). It is thought that this conversion occurs as a result of interaction between PrPC and PrPSc, which serves as a template to induce conformational change in PrPC from a predominately alpha-helical conformation to a predominately beta-sheet structure. This change is associated with the carboxy-terminal of PrPSc becoming protease-resistant. A minor, yet critical, difference exists between normal and disease-associated conformers; thus, discrimination between the forms is difficult, making detection of prion disease problematic. Currently, diagnostic tests for prion disease rely heavily on demonstration of protease-resistant forms of prion protein or the uncovering of hidden epitopes. In a Fiscal Year 2002 Department of Defense National Prion Research Program Investigator-Initiated Research Award, Dr. Man-Sun Sy proposed to develop an improved enzyme-linked immunosorbent assay (ELISA) for the diagnosis of TSEs. Because prion proteins dimerize, or form aggregates, Dr. Sy reasoned that certain epitopes would become inaccessible to antibody-binding, while concurrently presenting other epitopes in multiples, depending upon the composition of the aggregate. Dr. Sy concluded that if the epitope were present more than once, the same monoclonal antibody could be used as both capture and detection antibodies in the ELISA. After confirming the presence of dimers in recombinant human, mouse, ovid, and bovine prion protein preparations, 30 anti-PrP monoclonal antibodies were screened for their ability to recognize recombinant mouse PrP dimers (rMo-PrP). Five antibodies, four of which also recognized recombinant dimers from the other species, were found to react strongly, suggesting common features in their central regions, where the antibodies bind. An ELISA employing these four antibodies revealed that the four reacted strongly and with specificity against rMo-PrP dimers. Another ELISA found four antibodies that strongly recognized PrP in brain homogenates from infected mice. Only one antibody, 11G5, whose epitope is amino acids 114-130, was positive in both ELISAs, suggesting differences between in vitro and in vivo dimers. Additionally, it was found that the ELISA worked for three differing PrP strains and predominately recognized PrPSc aggregates of varying sizes from infected mice. The enhanced sensitivity was exemplified by detection of PrPSc 70 days post-infection, whereas previous assays required an incubation time of 140 days. Dr. Sy extrapolates that the ELISA has the potential to detect between 0.06 µg and 0.006 µg of aggregated PrP. ELISA results also imply a change in the helix 1 region of PrP through conversion of PrPC to PrPSc and possible interaction between the amino and carboxy-terminals of recombinant human PrP (1). Dr. Sy and colleagues have further refined the assay, combining an in vitro amplification step, the aggregation-specific ELISA, and a fluorescent amplification catalyzed by T7 RNA polymerase. The resulting assay, termed Am-A-FACCT, was found to have 100% specificity and sensitivity in detecting disease-associated prion aggregates in blood of infected mice at a late, but asymptomatic, stage. Furthermore, Am-A-FACCT was shown to have 50% sensitivity and 100% specificity at a very early time point following infection. Importantly, the assay was able to detect prion aggregates in the blood of mule deer with the naturally occurring prion disease, chronic wasting disease, implicating its potential use in safeguarding the food supply and preventing human contagion (2).
Publications:

Pan T, Chang B, Wong P, et al. 2005. An aggregation-specific enzyme-linked immunosorbent assay: Detection of conformational differences between recombinant PrP protein dimers and PrPSc aggregates. Journal of Virology 79(19):12355-12364.

Chang B, Cheng X, Yin S, et al. 2006. A blood test for prion: Disease associated prion aggregate is detected in the blood of infected but asymptomatic animals. Clinical Vaccine Immunology 2006 Nov 1; [Epub ahead of print].
 

bse-tester

Well-known member
Merry Christmas Mike and the best for the coming year to you and yours.

Now you know why we are using Case Western to validate our test for all known TSE's.

Got to run Mike, Christmas is a huge deal in our house and if "she who must be obeyed" catches me online, I am toast.

Merry Christmas.

Ron.
 

PORKER

Well-known member
Bad News for Venison Eaters--Mad Cow-Like Disease Among Deer is Highly Infectious
Study sounds CWD alarm
CSU scientists find disease can spread in deer saliva, blood
By Bill Scanlon
Rocky Mountain News, October 6, 2006
Straight to the Source
Deer can spread chronic wasting disease through saliva and blood, which means no part of an infected animal can be considered safe to eat, Colorado researchers discovered.

Up to now, there has been a widespread feeling that as long as the brain or lymph nodes were avoided, the rest of the deer was safe to eat.

The results of the study led by Colorado State University scientists will be reported in today's edition of Science.

Some herds in Colorado have no cases of CWD, and others have very low incidence. But among some herds around Fort Collins and Rocky Mountain National Park, the infection rate is as high as 6 percent.

And because deer with the disease, which causes them to stagger and lose weight, make themselves more frequent targets of hunters, chances can be quite good that the deer a hunter kills in an area with a high infection rate has CWD.

The first of Colorado's rifle seasons for deer and elk begins Oct. 14.

While there is no proof that humans can get CWD from eating the flesh of an infected deer, such cross-species transfer has been seen in a related prion-type illness - mad cow disease.

Ed Hoover, CSU microbiology professor and principal scientist for the study, suggests that hunters pay strict attention to the advice from the Colorado Division of Wildlife: Don't eat any part of a deer until its brain and lymph nodes have been tested for the presence of the abnormal prions that signal CWD.

Hunters also should wear gloves when they dress an animal to guard against accidental infection.

The finding that saliva can provide a pathway for the disease means CWD can spread easier than scientists previously thought, Hoover said.

Grooming and nibbling are common social behavior among deer. A diseased deer may chew on the hair of a noninfected deer. Later, when the healthy deer grooms itself, it can get infected through the saliva on the hair.

The CSU team used about a dozen deer that were kept indoors to ensure they couldn't get CWD from any source but the variables used in the study.

One group of deer were given the saliva of known infected deer to ingest. Parts of their tonsils were then clipped and tested for the presence of the abnormal prions that comprise CWD. Some had the disease in three months, others got it within a year.

A second group injected with the blood of infected deer also developed the disease. That means, according to researchers, that CWD can spread throughout a deer's body and throughout its flesh, albeit in smaller concentrations than in the brain and tonsils.

It also means that CWD possibly could be transferred through the bite of a mosquito.

Hoover's group already has embarked on a follow-up study.

In the hunt

• The first of Colorado's rifle seasons for deer and elk begins on Oct. 14.

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Copyright 2006, Rocky Mountain News. All Rights Reserved.
 
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