Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-07-09
2003-09-09
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000
Reexamination Certificate
active
06617119
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to immunoassays. More particularly the invention relates to an assay which allows for detection of a specific strain of a disease related conformational form of a protein (such as PrP
Sc
) which may have very low antibody binding affinity.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.
A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. (1982)
Science
218:1309-1311; Prusiner, Bolton et al. (1982)
Biochemistry
21:6942-6950; McKinley, Bolton et al. (1983)
Cell
35:57-62]. 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, Oesch et al. (1986)
Cell
46:417-428] and when PrP
C
is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases results from the transformation of the normal form of prion protein (PrP
C
) into the abnormal form (PrP
Sc
). There is no detectable difference in the amino acid sequence of the two forms. However, PrP
Sc
when compared with PrP
C
has a conformation with higher &bgr;-sheet and lower &agr;-helix content [Pan, Baldwin et al. (1993)
Proc Natl Acad Sci USA
90:10962-10966; Safar, Roller et al. (1993)
J Biol Chem
268:20276-20284]. The presence of the abnormal PrP
Sc
form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrP
Sc
plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103-143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991)
Curr Top Microbiol Immunol
172:21-38]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977)
Science
197:943-960; Medori, Tritschler et al. (1992)
N Engl J Med
326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
Prions exist in multiple isolates (strains) with distinct biological characteristics when these different strains infect in genetically identical hosts [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 165-186]. The strains differ by incubation time, by topology of accumulation of PrP
Sc
protein, and in some cases also by distribution and characteristics of brain pathology [DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6th Edition:235-280]. Because PrP
Sc
is the major, and very probably the only component of prions, the existence of prion strains has posed a conundrum as to how biological information can be enciphered in a molecule other than one comprised of nucleic acids. The partial proteolytic treatment of brain homogenates containing some prion isolates has been found to generate peptides with slightly different electrophoretic mobilities [Bessen and Marsh (1992) J Virol 66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling, Parchi et al. (1996) Science 274:2079-2082]. These findings suggested different proteolytic cleavage sites due to the different conformation of PrP
Sc
molecules in different strains of prions. Alternatively, the observed differences could be explained by formation of different complexes with other molecules, forming distinct cleavage sites in PrP
Sc
in different strains [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343:413-414]. Some researchers have proposed that different prion isolates may differ in the glycosylation patterns of prion protein [Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al. (1997) Lancet 349:99-100]. However, the reliability of both glycosylation and peptide mapping patterns in diagnostics of multiple prion strains is currently still debated [Collings, Hill et al. (1997) Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].
A system for detecting PrP
Sc
by enhancing immunoreactivity after denaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1, Ja 1990. Sufficiently sensitive and specific direct assay for infectious PrP
Sc
in biological samples could potentially abolish the need for animal inoculations completely. Unfortunately, such does not appear to be possible with current PrP
Sc
assays—it is estimated that the current sensitivity limit of proteinase-K and Western blot-based PrP
Sc
detection is in a range of 1 &mgr;g/ml which corresponds to 10
4
-10
5
prion infectious units. Additionally, the specificity of the traditional proteinase-K-based assays for PrP
Sc
is in question in light of recent findings of only relative or no proteinase-K resistance of undoubtedly infectious prion preparations [Hsiao, Groth et al. (1994)
Proc Natl Acad Sci USA
91:9126-9130] Telling, et al. (1996)
Genes
&
Dev.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly &bgr;-sheet structured units, and serves as a transporter of hormone thyroxine. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [Kelly (1996)
Curr Opin Strut Biol
1):11-7]. The cause of amyloid formation in FAP are point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in biopsy material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993)
Biochemistry
32(45):12119-27; McCutchen and Kelly (1993)
Biochem Biophys Res Commun
197(2) 415-21]. The mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear &bgr;-sheet structured amyloid fibrils [Lai, Colon et al. (1996)
Biochemistry
35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxine or triiodophenol [Miroy, Lai et al. (1996)
Proc Natl Acad Sci USA
93(26): 15051-6].
In view of the above points, there is clearly a need for a specific, high flow-through, and cost-effective assay for testing sample materials for the presence of specific strains of a pathogenic protein including transthyretin and prion protein.
SUMMARY OF THE INVENTION
Assay methodology of the invention allows for: (1) determining if a sample contains a conformation of a protein which is associated with disease and the concentration and amount of such if present; (2) determining the amount of a chemical compound such as a protease resistant disease related protein in a sample and by subtracting that amount from the total amount of disease related protein present determining the amount of protease sensitive disease protein in the sample; and (3) determining the strain and incubation time of a disease related protein by (i) relating the relative amounts of protease resistant and protease sensitive protein to known strains to thereby determine the strain; and (ii) pl
Cohen Fred E.
Prusiner Stanley B.
Safar Jiri G.
Bozicevic Karl
Bozicevic Field & Francis LLP
Housel James
Parkin Jeffrey S.
The Regents of the University of California
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