Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-08-23
2004-07-20
Winkler, Ulrike (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S139100, C424S141100, C435S007100, C435S326000, C435S331000, C530S388850
Reexamination Certificate
active
06765088
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to monoclonal antibodies reacting with certain epitopes of recombinant bovine prion protein, native and denatured normal or disease-specific prion proteins in soluble or insoluble state, stable hybridoma cell lines producing these monoclonal antibodies, recombinant expression vectors for the expression of recombinant bovine prion protein, purified recombinant bovine prion protein, a test kit for the diagnosis of prion diseases, diagnostic methods for the immunological detection of prion diseases, pharmaceutical preparations for the prevention and therapy of prion diseases, a method for clearing biological material from infectious prion proteins, and methods for the production of these materials.
Abbreviations used hereinbefore and hereinafter are the following:
BSA
bovine serum albumin
BSE
bovine spongiform encephalopathy
CSF
cerebrospinal fluid
CJD
Creutzfeldt-Jakob disease,
ECL
enhanced chemiluminescence
EDTA
ethylenediaminetetraacetic acid
ELIFA
enzyme linked immuno filtration assay
ELISA
enzyme linked immuno sorbent assay
Fab
fragment of antibody digested with papain
(Fab′)2
fragment of antibody digested with pepsin
FFI
Fatal Familial Insomnia
GP1-anchor
glycolipid-anchor which “ties” PrP to the outside of the
cell membrane
GSS
Gerstmann-Sträussler-Scheinker disease
H(A)T-
hypoxyanthine-(aminopterin)-thymidine medium
medium
HEPES
hydroxyethyl-piperazineethane sulfonic acid
HPLC
high performance liquid chromatography
IgG
immunoglobulin G
IPTG
isopropyl-&bgr;-D-thiogalactoside
mAB
monoclonal antibody
MOPS
morpholinepropanesulfonic acid
NC
nitrocellulose membrane
o
overnight
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
prion
proteinaceous infectious particle; the infectious
agent of prion diseases, supposedly consisting at least of
PrP
Sc
and maybe another yet unknown molecule
PrP
prion protein; refers to the common amino acid sequence
rather than to a distinct conformation of the two prion
protein isoforms
PrP
0/0
-mice
mice lacking a functional PrP gene
PrP
C
a normal host prion protein of unknown function; apparent
molecular weight 33-35 kDa, same amino acid chain,
and same glycosylation at two asparagine residues as
PrP
Sc
, is after proteinase K treatment fully digested.
PrP
Sc
the disease-specific, abnormal isoform of PrP
C
, with the
same amino acid chain, apparent molecular weight 33-35
kDa, glycosylated at two asparagine residues, is after
proteinase K treatment shortened to a 27-30 kDa C-
terminal fragment. Species-specific PrP
Sc
isoforms
term: human PrP
Sc
(instead of PrP
CJD
), bovine
PrP
Sc
(instead of PrP
BSE
) etc
rbPrP
recombinant bovine prion protein (amino acids 25 to 242 of
the bovine PrP gene according to Goldmann et al. 1991;
with an additional N-terminal methionine)expressed in
E. coli
comprising the bovine PrP open reading frame
except for the N-terminal signal sequence and the
C-terminal GPI-anchor sequence; both are cleaved of
during cellular processing. Since this protein is
not glycosylated it has a molecular weight of 23 kD
RT
room temperature
SAF
scrapie-associated fibrils; same as rods, plaque-like
multimeric PrP
SC
aggregates
SDS
sodium dodecyl sulfate
TBST
Tris-buffered saline, Tween 20
TMB
tetramethylbenzidine
Prion diseases are transmissible neurodegenerative diseases of the central nervous system (for review see Prusiner, 1991). They can be transmitted, inherited or occur sporadically and are observed in animals (e.g. bovine spongiform encephalopathy [BSE] in cattle, scrapie in sheep) as well as in humans (Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru). Prion diseases have a characteristically long incubation period and, with the onset of clinical symptoms, lead to ataxia, dementia, psychiatric disturbances and sleeplessness before inevitable death occurs. Neuropathological changes include vacuolar degeneration of brain tissue, astrogliosis and amyloid plaque formation. In the infected subjects, neither a systemic immune response, nor an obvious specific immune response like antibody production of PrP has been observed (Kasper et al., 1982; Garfin et al., 1978) however, some unspecific activation of immune cells in the brain was reported (Williams et al., 1995; Williams et al., 1994).
The infectious agent appears to exist in a variety of strains, which cause distinct incubation times and histopathology (Bruce et al., 1994; Hecker et al., 1992). Transmission of prion diseases is possible between species and most easily within the same species (Prusiner, 1991).
The infectious agent, the prion, is associated with a disease-specific protein, PrP
Sc
, that is an abnormal isoform of a host protein, PrP
C
(Oesch et al., 1985, Basler et al., 1986). Both, PrP
Sc
and PrP
C
, have an apparent molecular weight of 33-35 kDa on SDS-polyacrylamide gels. They have the same amino acid sequence and are glycosylated at two asparagine residues (Oesch et al., 1985) After proteinase K treatment, PrP
Sc
is shortened to a characteristic 27-30 kDa fragment while PrP
C
is fully digested (Bolton et al., 1982, Oesch et al., 1985), this led to the conclusion that the disease-specific isoform PrP
Sc
is partially protease resistant while the normal host isoform PrP
C
is not.
Studies on the synthesis and localization of the two PrP isoforms in cultured cells have shown that PrP
C
is attached to the cell surface by a glycosyl phosphatidylinositol (GPI) anchor while PrP
Sc
accumulates intracellularly within cytoplasmic vesicles (Stahl et al., 1987). Another difference between PrP
C
and PrP
Sc
is reflected in their three-dimensional structure PrP
Sc
has less alpha helical secondary structures and increased beta sheet content as compared to PrP
C
(Pan et al., 1993). So far, no chemical differences between the two isoforms have been observed (Stahl et al., 1993). In summary, PrP
Sc
and PrP
C
have the same amino acid sequence but a different folding. The misfolded prion protein is associated with infectivity and neurotoxicity.
The infectious agent is inactivated by treatments which denature proteins while reagents destroying nucleic acids have no effect (Diener et al., 1982; Alper et al., 1978). In addition, no single nucleic acid capable for coding a protein has been purified until date (Riesner et al., 1993). This has lead to the hypothesis that PrP
Sc
itself might comprise the infectious particle (Griffith, 1967; Prusiner, 1982). According to this hypothesis, replication of infectivity is achieved by the replication of the pathogenic conformation. It is supposed that infectious PrP
Sc
molecules convert the normal host protein PrP
C
to the PrP
Sc
conformation (Cohen et al., 1994). Conversion of PrP
C
to PrP
Sc
was claimed to have been achieved in vitro thereby mimicking species and strain characteristics comparable to the conversion dynamics in vivo (Kocisko et al., 1994; Bessen et al., 1995). However, these in vitro converted PrP
Sc
molecules have, to date, not shown to be infectious.
The function of the normal host protein, PrP
C
, is unknown. Mice devoid of PrP
C
are viable and show no obvious signs of neurological and physical impairment (Bueler et al., 1992). In addition, these mice are not susceptible to infection with prions, underlining the central importance of PrP in the replication of infectivity and/or pathology of these diseases (Bueler et al., 1993; Prusiner et al., 1993). More subtle investigations of PrP knockout mice revealed impaired synaptic function (Collinge et al., 1994) and altered sleep regulation (Tobler et al., 1996). However, a molecular function of PrP
C
could not be deduced from these findings.
Prion diseases have gained public interest with the appearance of BSE in the early eighties in Great Britain (Hope et al., 1988); for review see (Wells and Wilesmith, 1995). The disease is supposed to have been transmitted by feeding prion-contaminated meat and bone meal to cattle. It is thought that BSE prions originated from scrapie-diseased sheep by crossing the species barrier from sheep
Korth Carsten
Moser Markus
Oesch Bruno
Stierli Beat
Stregt Peter
Sidley Austin Brown & Wood LLP
Universität Zürich
Winkler Ulrike
LandOfFree
Immunological detection of 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 Immunological detection of prions, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Immunological detection of prions will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3239713