Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten
Reexamination Certificate
2000-10-27
2003-09-16
Scheiner, Laurie (Department: 1648)
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
C436S536000, C435S005000, C435S007100
Reexamination Certificate
active
06620629
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods of detecting prions in samples such as blood or tissue.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects a first species of animal (e.g., a human) will not infect a second species which is genetically diverse form the first species (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (“PrP”) (Bolton et al.,
Science
218:1309-11 (1982); Prusiner et al.,
Biochemistry
21:6942-50 (1982); McKinley et al.,
Cell
35:57-62 (1983)). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP
C
is encoded by a single-copy host gene (Basler et al.,
Cell
46:417-28 (1986)) and is normally found at the outer surface of neurons. During a post-translational process, PrP
Sc
is formed from the normal, cellular PrP isoform (PrP
C
), and prion diseases result from conversion of PrP
C
into a modified isoform called PrP
Sc
. PrP
Sc
is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans.
See Prusiner, S. B.,
Science
252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats, chronic wasting disease of deer and elk, and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith, J. and Wells,
Microbiol. Immunol
. 172:21-38 (1991)). Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) (Gajdusek, D. C.,
Science
197:943-960 (1977); Medori et al.,
N. Engl. J Med
. 326:444-449 (1992)). The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Variations in prions, which cause different disease phenotypes, are often referred to as strains. Each prion strain produces a specific phenotype of prion disease as manifested by the length of the incubation time, the topology of PrP
Sc
accumulation, and the distribution of pathological lesions (Fraser and Dickinson 1968; Fraser and Dickinson 1973; Bruce, McBride et al. 1989; Taraboulos, Jendroska et al. 1992; DeArmond, Yang et al. 1993; Scott, Groth et al. 1997). Strains replicate with a high degree of fidelity, which demands a mechanism that can account for this phenomenon. That strains could be accounted for because PrP
Sc
might exist in multiple conformations was postulated, but supporting evidence was initially lacking (Prusiner 1991; Cohen, Pan et al. 1994). Subsequently, different lines of investigation, one on the isolation of prion strains from mink by passage in hamsters (Bessen and Marsh 1994) and the other on the passage of inherited human prion diseases to transgenic (Tg) mice (Telling, Parchi et al. 1996), converged to argue that the properties of prion strains are enciphered in the conformation of PrP
Sc
.
The detection of PrP
Sc
in biological products is of critical importance, as prion diseases are transmissible. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts (Brown et al.,
Lancet
340:24-27 (1992)). In addition, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism (Alpers, M. P.,
Slow Transmissible Diseases of the Nervous System
Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90 (1979)). Numerous young adults treated with HGH derived from human pituitaries have developed CJD (Koch et al.,
N. Engl. J Med
. 313:731-733 (1985); Brown et al.,
Lancet
340:24-27 (1992); Fradkin et al.,
JAMA
265:880-884 (1991); Buchanan et al.,
Br. Med. J
. 302:824-828 (1991)). In addition, hundreds of children in France have been treated with growth hormone extracted from dead bodies at the risk of developing CJD (see
New Scientist
, Nov. 20, 1993, page 10.) That the HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH (Gibbs, Jr. et al.,
N. Engl. J. Med
. 328:358-359 (1993)).
The long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD for decades in thousands of people treated with HGH worldwide. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotrophin hormone (Healy et al.,
Br. J. Med
. 307:517-518 (1993); Cochius et al.,
Aust. N. Z. J. Med
. 20:592-593 (1990); Cochius et al.,
J. Neurol. Neurosurg. Psychiatry
55:1094-1095 (1992)) as well as at least 11 patients receiving dura mater grafts (Nisbet et al.,
J. Am. Med. Assoc
. 261:1118 (1989); Thadani et al.,
J. Neurosurg
. 69:766-769 (1988); Willison et al.,
J. Neurosurg. Psychiatric
54:940 (1991); Brown et al.,
Lancet
340:24-27 (1992)). These cases of iatrogenic CJD underscore the need for screening pharmaceuticals and biological products that might possibly be contaminated with prions.
The importance of detecting prions in biological products has been heightened by the possibility that bovine prions have been transmitted to humans who developed new variant Creutzfeldt-Jakob disease (nvCJD) (G. Chazot et al.,
Lancet
347:1181 (1996); R. G. Will et al.
Lancet
347:921-925 (1996)). Earlier studies had shown that the N-terminus of PrP
Sc
could be truncated without loss of Scrapie infectivity (S. B. Prusiner et al.,
Biochemistry
21:6942-6950 (1982); S. B. Prusiner et al.,
Cell
38:127-134 (1984)) and correspondingly, the truncation of the N-terminus of PrP
Sc
still allowed its conversion into PrP
Sc
(M. Rogers et al.,
Proc. Natl. Acad. Sci. USA
90:3182-3186 (1993)). The ability of transmission of nvCJD from cattle to humans has been confirmed through in vivo testing, suggesting that the December 20 issue of Proceedings of National Academy of Sciences undermining the comforting presumption that the documented “species barrier” is relevant to this new strain (M. R. Scott et al.,
Proc. Natl. Acad. Sci. USA
96:15137-15142 (1999)).
In view of such, there clearly is a need for a convenient, cost-effective means for identifying prions which cause CJD in biological products, e.g., blood and blood products.
SUMMARY OF THE INVENTION
The present invention provides assays that can identify the levels of both protease sensitive and protease resistant conformers of PrP
Sc
in a sample. In a preferred embodiment, the assay comprises the steps of 1) determining levels of total PrP
Sc
in a sample; 2) subjecting the PrP
Sc
fraction to treatment with a protease that selectively hydrolzes the protease sensitive PrP
Sc
(sPrP
Sc
) conformers and 3) quantifying the levels of sPrP
Sc
in the sample. The ability to detect sPrP
Sc
allows early detection of prions, since the PrP
Sc
in easily accessible biological samples such as blood is predominantly sPrP
Sc
and not protease resistant PrP
Sc
, i.e., not rPrP
Sc
. These assay can identify prion infection in a sample prior to the accumulation of the protease resistant PrP
Sc
(rPrP
Sc
) conformers to a level associated with classic symptoms of prison infection. The present invention thus provides an improved method of early detection of these infectious agents in products such as human biologicals and food.
Methods of the present invention also have the ability to distinguish between different prion isolates (“strains”), and thus can identify a specific prion strain in an infected sample. The ratio of rPrP
Prusiner Stanley B.
Safar Jiri
Bozicevic Karl
Bozicevic Field & Francis LLP
Parkin Jeffrey S.
Scheiner Laurie
The Regents of the University of California
LandOfFree
Method for detecting prions does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for detecting prions, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for detecting prions will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3049165