U.S. patent application number 15/665323 was filed with the patent office on 2018-06-21 for detection of infectious prion protein by seeded conversion of recombinant prion protein.
This patent application is currently assigned to THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Serv, THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Ryuichiro Atarashi, Byron W. Caughey, Roger A. Moore.
Application Number | 20180172709 15/665323 |
Document ID | / |
Family ID | 39832401 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180172709 |
Kind Code |
A1 |
Caughey; Byron W. ; et
al. |
June 21, 2018 |
DETECTION OF INFECTIOUS PRION PROTEIN BY SEEDED CONVERSION OF
RECOMBINANT PRION PROTEIN
Abstract
The present disclosure relates to methods and compositions for
the detection of infectious proteins or prions in samples,
including the diagnosis of prion related diseases. One embodiment
is an ultrasensitive method for detecting PrP-res (PrP.sup.Sc) that
allows the use of recombinant PrP-sen (rPrP-sen) as a substrate for
seeded polymerization. A sample is mixed with purified rPrP-sen to
make a reaction mix which is incubated to permit aggregation of the
rPrP-sen with the PrP-res that may be present in the sample. Any
aggregates are intermittently disaggregated by agitation and the
reaction allowed to proceed to amplify target substrate. Any
rPrP-res.sup.(Sc) in the reaction mix is detected to indicate the
presence of PrP-res in the original sample. In the QUIC method in,
the reaction mixture is shaken intermittently. The surprising speed
and efficiency of the method permits the rapid identification and
diagnosis of prion disease.
Inventors: |
Caughey; Byron W.;
(Hamilton, MT) ; Atarashi; Ryuichiro; (Nagasaki,
JP) ; Moore; Roger A.; (Hamilton, MT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
THE UNITED STATES OF AMERICA, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
39832401 |
Appl. No.: |
15/665323 |
Filed: |
July 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14263703 |
Apr 28, 2014 |
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15665323 |
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13489321 |
Jun 5, 2012 |
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14263703 |
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12177012 |
Jul 21, 2008 |
8216788 |
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13489321 |
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61021865 |
Jan 17, 2008 |
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60961364 |
Jul 20, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6896 20130101;
G01N 2800/28 20130101; G01N 2800/2828 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for detecting protease resistant prion protein
(PrP-res) in a sample comprising: (a) mixing the sample with a
purified recombinant protease-sensitive prion protein (rPrP-sen) to
make a reaction mix, wherein the rPrP-sen comprises the amino acid
sequence set forth as SEQ ID NO: 3, the amino acid sequence set
forth as SEQ ID NO: 4, or the amino acid sequence set forth as
amino acids 23-231 of SEQ ID NO: 8; (b) performing an amplification
reaction comprising: (i) incubating the reaction mix at about 37 to
55.degree. C. to permit coaggregation of the rPrP-sen with the
PrP-res that may be present in the reaction mix, and maintaining
incubation conditions that promote coaggregation of the rPrP-sen
with the PrP-res and result in a conversion of the rPrP-sen to the
recombinant protease resistant prion protein initiated by the
presence of prions (rPrP-res.sup.(Sc), which is initiated by the
presence of PrP-res in the sample, while inhibiting development of
spontaneously occurring recombinant prion protein
(rPrP-res.sup.(spon)); (ii) agitating aggregates formed during step
(i), in shaking cycles, wherein each shaking cycle of the shaking
cycles comprises a period of rest and a period of shaking, and
wherein the period of rest is about 30 seconds in length and the
period of shaking is about 30 seconds in length, or wherein the
period of rest is about 60 seconds in length and the period of
shaking is about 60 seconds in length, wherein agitating is
performed for about 2 to 48 hours in the absence of sonication; and
(c) detecting rPrP-res.sup.(Sc) in the reaction mix, wherein
detection of rPrP-res.sup.(Sc) in the reaction mix indicates that
PrP-res was present in the sample.
2. The method of claim 1, wherein detecting the rPrP-res.sup.(Sc)
comprises detecting rPrP-res.sup.(Sc) aggregates in the sample.
3. The method of claim 1, wherein the method further comprises
digesting the reaction mix with proteinase K prior to detecting
rPrP-res.sup.(Sc) in the reaction mix.
4. The method of claim 2, wherein detecting rPrP-res.sup.(Sc)
comprises detecting rPrP-res.sup.(Sc) with an antibody that
specifically binds to prion protein.
5. The method of claim 4, wherein the antibody is a polyclonal
antibody.
6. The method of claim 4, wherein the antibody is a monoclonal
antibody.
7. The method of claim 1, wherein steps (a) and (b) are repeated
for approximately 48 hours.
8. The method of claim 1, wherein the reaction mix is incubated at
45.degree. C. to 55.degree. C.
9. The method of claim 1, wherein the reaction mix is incubated at
45.degree. C.
10. The method of claim 1, wherein the rPrP-sen consists of the
amino acid sequence set forth as one of SEQ ID NO: 3, SEQ ID NO: 4,
amino acids 23-231 of SEQ ID NO: 8, or SEQ ID NO: 8.
11. The method of claim 1, wherein the sample is a tissue sample
from an animal.
12. The method of claim 1, wherein prion can be detected in a
sample containing no more than about 1 fg PrP-res.
13. The method of claim 1, wherein the method is a method of
diagnosing a prion disease.
14. The method of claim 1, wherein detecting rPrP-res.sup.(Sc) in
the reaction mix comprises the use of a fluorescence assay.
15. A method for amplifying and detecting the human protease
resistant prion protein (PrP-res) in a sample comprising: (a)
mixing the sample with purified recombinant protease-sensitive
prion protein (rPrP-sen) comprising the amino acid sequence forth
as SEQ ID NO: 8, SEQ ID NO: 3 or SEQ ID NO: 4 to make a reaction
mix; (b) performing an amplification reaction comprising: (i)
incubating the reaction mix at about 37.degree. C. to 55.degree. C.
to permit formation of aggregates of the rPrP-sen with the human
PrP-res that may be present in the reaction mix, and maintaining
incubation conditions that promote aggregation of the rPrP-sen with
the human PrP-res and result in a conversion of the rPrP-sen to
recombinant protease resistant prion protein initiated by the
presence of prions (rPrP-res.sup.(Sc) while inhibiting development
of spontaneously occurring recombinant prion protein
(rPrP-res.sup.(spon)); (ii) agitating aggregates formed during step
(i), in shaking cycles, wherein each shaking cycle of the shaking
cycles comprises an equal period of rest and a period of shaking,
and wherein the shaking cycle is from about 60 to 120 seconds in
length, and wherein agitating is performed in the absence of
sonication; (c) detecting rPrP-res.sup.(Sc) in the reaction mix
using fluorescence, wherein detection of rPrP-res.sup.(Sc) in the
reaction mix indicates that PrP-res was present in the sample.
16. The method of claim 15, wherein the purified recombinant
protease-sensitive prion protein (rPrP-sen) comprises hamster
rPrP-sen and human rPrP-sen.
17. The method of claim 15, comprising incubating the reaction mix
at about 37.degree. C., 45.degree. C. or 55.degree. C.
18. The method of claim 15, wherein the purified recombinant
protease-sensitive prion protein (rPrP-sen) consists of the amino
acid sequence set forth as SEQ ID NO: 8, the amino acid sequence
set forth as amino acids 23-231 of SEQ ID NO: 8, the amino acid
sequence set forth as SEQ ID NO: 3 or the amino acid sequence set
forth as SEQ ID NO: 4.
19. The method of claim 15, wherein agitating aggregates formed
during step (i), in shaking cycles, is performed for about 2 to 48
hours.
20. A method for detecting Creutzfeldt-Jakob disease in a subject,
comprising: (a) mixing a biological sample from the subject with
purified recombinant protease-sensitive prion protein (rPrP-sen)
comprising the amino acid sequence forth as amino acids 23-231 of
SEQ ID NO: 8 to make a reaction mix; (b) performing an
amplification reaction comprising: (i) incubating the reaction mix
at about 37.degree. C. to 55.degree. C. to permit formation of
aggregates of the rPrP-sen with any human protease resistant prion
protein (PrP-res) that may be present in the reaction mix, and
maintaining incubation conditions that promote aggregation of the
rPrP-sen with the human PrP-res and result in a conversion of the
rPrP-sen to recombinant protease resistant prion protein initiated
by the presence of prions (rPrP-res.sup.(Sc)) while inhibiting
development of spontaneously occurring recombinant prion protein
(rPrP-res.sup.(spon)); (ii) agitating aggregates formed during step
(i), in shaking cycles, wherein each shaking cycle of the shaking
cycles comprises an equal period of rest and a period of shaking,
and wherein the shaking cycle is about 120 seconds in length, and
wherein agitating is performed in the absence of sonication; (c)
detecting rPrP-res.sup.(Sc) in the reaction mix using fluorescence,
wherein detection of rPrP-res.sup.(Sc) in the reaction mix
indicates that the subject has Cruzefeld-Jakob disease.
21. The method of claim 20, wherein the sample is a blood sample or
a cerebral spinal fluid sample.
Description
PRIORITY
[0001] This is a continuation of U.S. patent application Ser. No.
14/263,703, filed on Apr. 28, 2014, now abandoned, which is a
continuation of U.S. patent application Ser. No. 13/489,321, filed
on Jun. 5, 2012, now abandoned, which is a continuation of U.S.
patent application Ser. No. 12/177,012, filed on Jul. 21, 2008, now
issued as U.S. Pat. No. 8,216,788, which claims the benefit of U.S.
Provisional Application No. 61/021,865, filed Jan. 17, 2008, and
U.S. Provisional Application 60/961,364, filed Jul. 20, 2007. All
of these prior applications are incorporated herein by reference in
their entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to methods and compositions
for the detection of infectious proteins or prions in samples,
including the diagnosis of prion related diseases.
BACKGROUND
[0003] Prion diseases, which are also called transmissible
spongiform encephalopathies (TSEs), include a group of fatal
infectious neurodegenerative diseases that include
Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler
Scheinker syndrome (GSS), fatal familial insomnia (FFI) and
sporadic fatal insomnia (sFI) in humans, and scrapie, bovine
spongiform encephalopathy (BSE) and chronic wasting disease (CWD)
in animals. These diseases are characterized by brain vacuolation,
astrogliosis, neuronal apoptosis, and the accumulation of misfolded
prion protein (PrP-res, also known as PrP.sup.Sc and PrP.sup.CJD)
in the central nervous system. TSEs have incubation periods of
months to years, but after the appearance of clinical signs they
are rapidly progressive, untreatable, and invariably fatal.
Attempts at TSE risk reduction have led to profound changes in the
production and trade of agricultural goods, medicines, cosmetics,
and biotechnology products.
[0004] The hallmark event of prion disease is the formation of an
abnormally folded protein called PrP.sup.Sc (or PrP-res), which is
a post-translationally modified version of a normal protein, termed
PrP.sup.C (also known as PrP-sen). A prion detection method termed
protein misfolding cyclic amplification (PMCA) is based on the
ability of prions to replicate in vitro in cell lysates containing
PrP.sup.C (see, for instance, WO0204954). However, the limitations
of PMCA include the time required to achieve optimal sensitivity
(.about.3 weeks) and the requirement for brain-derived PrP-sen as
the amplification substrate.
[0005] Castilla et al., Methods in Enzymology 412:3-21 (2006) has
stated that it has not been possible to use PMCA with highly
purified prion proteins such as PrP.sup.C. Although the reason for
this limitation was unknown, it was believed that factors in brain
homogenates were needed to catalyze prion propagation. Recombinant
PrP-sen expressed from E. coli also lacks glycosylation and the
glycophosphatidylinositol (GPI) anchor, which was additionally
believed to contribute to the difficulty of using rPrP-sen in
amplification reactions. Such rPrP-sen has been converted to
protease-resistant forms with very limited yields when mixed with
PrP.sup.Sc in the past.
[0006] Another problem with PMCA is that the formation of
PrP.sup.Sc reaches a plateau as the number of amplification cycles
increases. Castrillon et al. (US Patent Publication No.
2006/0263767) attempted to overcome this problem by serial
amplification of prion protein by removing a portion of the
reaction mix and incubating it with additional non-pathogenic
protein. Although serial amplification PMCA (saPMCA) increases
prion amplification and enhances the sensitivity of the assay, the
necessity of performing multiple rounds of serial amplification has
decreased the overall practicality of the process.
[0007] Supattapone and Deleault (PCT Publication No. WO
2007/082173) also note that efficiency of amplification may require
a cellular factor other than PrP-sen. They disclose in vitro
amplification of immunoaffinity or exchange chromatography purified
PrP-sen in the presence of RNA, synthetic polyanions and partially
purified substrates to increase the sensitivity of diagnostic
methods for detecting PrP-res.
[0008] However, there continues to be a need for a more rapid
method for the detection of PrP-res that is sensitive enough to
detect low level prion contamination. The widespread public health
concern about TSE diseases could be allayed by the development of
such a test.
SUMMARY OF THE DISCLOSURE
[0009] Disclosed herein is an ultrasensitive method for detecting
prion protein (for instance, PrP-res or PrP.sup.Sc) that allows the
use of recombinant PrP-sen (rPrP-sen) as a substrate for seeded
polymerization. These methods include the use of an rPrP-res
amplification assay, which includes methods such as rPrP-PMCA or
QUIC, which differ in the method used to agitate the reaction. The
rPrP-res amplification assays are surprisingly much faster than
existing PMCA methods, yet it still retains sufficient sensitivity
to detect extremely low levels of PrP-res. The surprising rapidity
of the method permits the rapid identification and diagnosis of
prion disease, which can limit the transmission of prion diseases,
particularly through the food supply.
[0010] One embodiment of the disclosure is a method for detecting
PrP-res (PrP.sup.Sc) in a sample. The method includes the steps of
(a) mixing the sample with purified rPrP-sen to make a reaction
mix, and (b) performing an amplification reaction between PrP-res
(PrP.sup.Sc) and rPrP-sen in the mixture that results in the
formation and amplification of one or more specific forms of
recombinant PrP-res (for instance, rPrP-res.sup.(Sc)). The
amplification reaction includes the steps of (i) incubating the
reaction mix to permit co-aggregation or co-polymerization of the
rPrP-sen with the PrP-res that may be present in the reaction mix,
and (ii) agitating any aggregates or multimers formed during step
(i), for instance by shaking or sonication, and (iii) repeating
steps (i) and (ii) one or more times. In step (i), aggregation of
the rPrP-sen with any PrP-res that may be present in the sample
results in a conversion of the rPrP-sen to rPrP-res.sup.(Sc). After
the amplification reaction is carried out, rPrP-res.sup.(Sc) is
detected in the reaction mix as an amplified indicator of any
PrP-res originally present in the sample. This amplification
procedure can be performed on an initial sample of interest, such
that the method is performed only as a single round of
amplification. Optionally, a serial amplification reaction is
carried out with the same steps as the first round reaction, except
an aliquot of the amplified reaction mixture (instead of the
original sample) is mixed with purified rPrP-sen.
[0011] In particular embodiments, the amplification reaction is
carried out under conditions that inhibit production of
spontaneously aggregated rPrP-res (rPrP-res.sup.(spon)) that is
independent of the presence of PrP-res in the sample, because that
by-product has surprisingly been found to interfere with the
desired aggregation reaction of rPrP with PrP-res and can
complicate the detection of rPrP-res.sup.(Sc). Inhibiting the
production of the by-product increases the speed, sensitivity, and
reliability of the amplification reaction.
[0012] In the embodiment referred to as the QUIC assay, agitation
of aggregates to disaggregate them is carried out in
multiple-container trays that are physically shaken without
sonication to transmit the disaggregating energy substantially
equally to all the containers in the tray. The use of shaking
instead of sonication has been found to provide a more easily
duplicated and rapid test that retains a high degree of
sensitivity.
[0013] The foregoing and other features and advantages of the
disclosure will become more apparent from the following detailed
description of several embodiments which proceeds with reference to
the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A-1D are a series of digital images of gels showing
the comparison of hamster proteinase K resistant prion protein
(HaPrP.sup.Sc)-seeded and unseeded recombinant hamster proteinase
K-sensitive prion protein (rHaPrP-sen) conversion reactions. FIG.
1A is a digital image of a gel comparing the results of the assay
when a designated amount of purified HaPrP.sup.Sc was incubated
with 0.2 mg/ml rHaPrP-sen in 0.1% sodium dodecyl sulphate (SDS) and
0.1% TX-100 in phosphate buffered saline (PBS) for 24 hours, with
(lanes 5-8) or without (lanes 1-4) periodic sonication (a 40-second
pulse every hour). rHaPrP-sen was omitted from reactions shown in
lanes 1 and 5. The reactions were digested with proteinase K (PK;
0.025:1 PK/rPrP weight/weight) and equivalent aliquots were
subjected to immunoblotting using the polyclonal antibody R20,
which was raised against prion protein residues 219-232. Open
circles and black diamonds mark the 17- and 10-kDa fragments,
respectively. FIG. 1B is a digital image of a gel showing the
results of the assay when aliquots of first round
HaPrP.sup.Sc-seeded, sonicated reaction products shown in lane 7 of
FIG. 1A were diluted by the designated factors into fresh
rHaPrP-sen and subjected to a second round of sonicated or
unsonicated reactions and PK treatments as in FIG. 1A. Lanes
designated "No seed" indicate reactions that were left unseeded.
FIG. 1C is a series of digital images of three gels showing the
antibody reactivity of PK-treated reaction products, which was
determined after three sequential rounds of reactions seeded in the
first round with 0 or 40 ng PrP.sup.Sc. The reactions were diluted
100-fold into fresh rHaPrP-sen between each round. The third round
reactions were digested with the designated PK:PrP ratios and
analyzed by immunoblot with D13, R18 and R20 antibodies. The
respective antibody epitopes are contained within the prion protein
residues indicated in parentheses. Lanes 1 and 5 show 2 .mu.l
samples (400 ng of rHaPrP) without PK digestion. Lane 9 is 100 ng
rHaPrP-sen without PK digestion. Asterisks indicate dimer formed
from 12-13 kDa fragments, suggested by their size and lack of
recognition by D13, an antibody which would react with full-length
rPrP but not with a dimer of 13-kDa fragments containing the
C-terminal epitope of R20. FIG. 1D is a digital image showing
silver staining of rHaPrP-res.sup.(Sc) or unseeded
(rHaPrP-res.sup.(spon)) third-round after PK digestion (0.025:1
PK/rPrP). Positions of molecular mass markers are designated in
kDa.
[0015] FIGS. 2A-2B are a pair of digital images of gels showing the
detection limits of rPrP-protein misfolding cyclic amplification
(rPrP-PMCA). FIG. 2A is a pair of digital images showing the
results of the first round of rPrP-PMCA. Serially diluted scrapie
brain homogenate (ScBH) containing the designated amounts of
PrP.sup.Sc was used as seeds. Normal brain homogenate (NBH) (1%)
was used for negative controls (lanes 8-10) and as a diluent for
the ScBH. The reactions seeded with 2-50 ag of PrP.sup.Sc or NBH
were done in triplicate. Untreated rHaPrP-sen is shown in lanes 1
and 11. All other samples were treated with PK (0.025:1 PK/rPrP
wt/wt ratio) for 1 hour at 37.degree. C. Samples were probed with
anti-PrP monoclonal antibody D13. FIG. 2B is a pair of digital
images showing the results of the second round of rPrP-PMCA. One
tenth volume (8 .mu.l) of the first round samples was transferred
to a newly prepared substrate mixture. PK digestion and
immunoblotting were done as described in Example 1. Similar results
were obtained in another independent experiment. Positions of
molecular mass markers are designated in kDa.
[0016] FIGS. 3A-3B are a pair of digital images of gels showing
seeding competition between rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon). Purified HaPrP.sup.Sc and
rHaPrP-res.sup.(spon) were each used to initiate three successive
rounds of rPrP-PMCA. Aliquots of the third-round reactions
containing similar amounts of either rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon) were used to seed fourth round reactions,
which were subjected to sonication cycles over 24 hours as
described in Example 2. The relative seed amounts of 1, 10 and 100
designate reactions seeded with 0.08, 0.8 or 8 .mu.l, respectively,
of the final third-round reaction volume. PK-treated reaction
products of the third-round (FIG. 3A) and fourth-round (FIG. 3B)
reactions were analyzed by immunoblotting with antiserum R20. The
17-kDa and 10-kDa bands specific for the rHaPrP-res.sup.(Sc)- and
rHaPrP-res.sup.(spon)-seeded reactions, respectively, are marked
with an open circle and a diamond, respectively. Positions of
molecular mass markers are designated in kDa.
[0017] FIGS. 4A-4B are a pair of digital images of gels showing the
results of seeding rPrP-PMCA with cerebrospinal fluid (CSF).
Aliquots (2 .mu.l) of CSF taken from normal hamsters (n=3) or
hamsters in the clinical phase of scrapie (n=6) were used to seed
rPrP-PMCA reactions Immunoblots of the PK-digested products of the
first 24-hour round are shown in FIG. 4A. Ten percent of each first
round reaction volume was used to seed a second 24-hour round of
rPrP-PMCA and the PK-digested products of the latter are shown in
FIG. 4B. Antisera D13 and R20 were used for the immunoblots. Lane 1
of each panel shows 100 ng HaPrP-sen without PK treatment. The
rPrP-PMCA reaction products were digested with a PK:PrP ratio of
0.025:1 (w/w). The positions of the 17-kDa rHaPrP-res.sup.(Sc) band
are marked with a circle.
[0018] FIGS. 5A-5B are a pair of digital images of gels and a graph
showing the generation of thioflavin-T positive, protease resistant
recombinant mouse prion protein (rMoPrP) fragments by sonication.
FIG. 5A is a pair of digital images of gels showing the results of
rPrP-PMCA. Solutions of rMoPrP (0.4 mg/ml, 16 .mu.M) in PBS pH 7.4,
and SDS (0-0.5%) were prepared in 100 .mu.L volumes. The tubes were
incubated at 37.degree. C. in a cuphorn sonicator bath. The samples
were then subjected to repeated cycles of 9 minutes of incubation
followed by 1 minute of sonication at 100% power. After 18 hours,
the samples were treated with PK. PK-digested samples were
immunoblotted with antibody R20. Upper and lower panels correspond
to incubations without and with sonication, respectively. Lane 1 of
each panel shows 100 ng of rHaPrP-sen without PK digestion.
Molecular mass markers are indicated in kilodaltons on the left
side. FIG. 5B is a graph showing the kinetics of increase in the
fluorescence of the amyloid stain thioflavin T when combined with
sonicated or unsonicated samples of rMoPrP in 0.1% SDS as in FIG.
5A. Thioflavin T (ThT) fluorescence typically increases upon
interaction with amyloid fibrils (Prusiner (1998) Proc. Natl. Acad.
Sci. U.S.A. 95, 13363-13383). Aliquots (5 .mu.l) were withdrawn at
each time point and diluted into 10 .mu.M thioflavin T, 50 mM
glycine pH 8.5 to a volume of 100 .mu.l. Fluorescence emission was
measured at 482 nm with excitation at 445 nm. Three independent
reactions with sonication are shown relative to a single control
reaction done without sonication.
[0019] FIGS. 6A-6C are a series of digital images of gels and
graphs showing the results of seeding reactions with sonicated
rPrP-res under unsonicated conditions. FIG. 6A is a pair of digital
images of immunoblots showing products of unsonicated conversion
reactions that were either unseeded or seeded with 1.6 .mu.l
aliquots of sonicated reactions containing rMoPrP-res(spon) ([Mo])
or rHaPrP-res(spon) ([Ha]) and total prion protein concentrations
of 0.4 mg/ml. The seed volumes were added to 80 .mu.L 0.4 mg/ml
rMoPrP-sen or rHaPrP-sen in 0.1% SDS, 10 .mu.M thioflavin T (ThT)
and PBS, pH 7.4, in 96-well assay plates. The reactions were
incubated for 96 hours without sonication. Aliquots were digested
with PK at the designated PK:rPrP ratio and analyzed by
immunoblotting with antibody R20. The first lane of each panel
shows 100 ng of rPrP-sen without PK treatment. FIG. 6B is a pair of
graphs showing the kinetics of reactions seeded with the designated
% volumes of rMoPrP-res(spon)-containing reaction products,
followed by monitoring ThT fluorescence at 482 nm (left graph; data
points are means.+-.SD, n=3). The results of heterologous reactions
in which rMoPrP-res.sup.(spon) was used to seed the conversion of
rHaPrP-sen are also shown. The right graph shows the linear
relationship between seed concentration and ThT fluorescence
(r2=0.998) after 32 hours under these unsonicated reaction
conditions. FIG. 6C is a pair of graphs showing the kinetics of
analogous homologous and heterologous reactions seeded with
rHaPrP-res(spon)-containing reaction products. The right graph
shows the linear relationship between the amount of seed and ThT
fluorescence after 8 hours (r2=0.997).
[0020] FIG. 7 is a pair of digital images of gels showing the
effects of SDS and Sarkosyl upon treatment of rPrP-PMCA reaction
products with high concentrations of PK. Aliquots of third round
PrPSc-seeded or unseeded rPrP-PMCA reaction products containing
either rHaPrP-res.sup.(Sc) (Sc) or rHaPrP-res.sup.(spon) (spon)
were treated with 20 .mu.g/ml PK (PK:PrP ratio=0.5:1) as described
in Example 1 except for the addition of the designated
concentrations of SDS or Sarkosyl. This PK concentration is 20-fold
higher than used in most of the other experiments described herein.
This stronger PK treatment in 0.1-2% SDS severely reduced the
relative recovery of the characteristic 17 kDa rHaPrP-res.sup.(Sc)
band (compare to FIG. 5B, lanes 2-7, for example). However, 1-2%
Sarkosyl strongly enhanced the recovery of the 17-kDa
rHaPrP-res.sup.(Sc) band while retaining striking differences
between the banding profiles of rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon). Therefore, the addition of Sarkosyl together
with higher concentrations of PK can provide rPrP-PMCA digestion
conditions that are more robust and less sensitive to minor
variations in PK activity or total protein concentrations of the
reaction mixtures.
[0021] FIGS. 8A-8F are a series of digital images of electron
micrographs showing the ultrastructure of rHaPrP-res.sup.(Sc)
(FIGS. 8A, 8C, 8E) and rHaPrP-res.sup.(spon) (FIGS. 8B, 8D, 8F). To
further characterize the structure of rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon), the samples were examined with transmission
electron microscopy. Electron micrographs of both samples prior to
PK digestion revealed thick overlapping fibre bundles, the
definition and edges of which were somewhat blurred (FIGS. 8A, 8B).
After PK digestion (FIGS. 8C, 8D), the fibrils within these bundles
were better resolved, indicating that the PK resistant cores of the
fibrils are coated with PK sensitive material, either the
rHaPrP-sen that has yet to convert to a more resistant structure,
the flexible N-termini projecting outwards, or both. In some
instances more separated fibrils could be detected, although their
tendencies to cluster together gave false impressions of increased
width when viewed without further magnification. Storing the
material in water further dissociated the bundles, yielding more
clearly defined fibril clusters for comparison (FIGS. 8E, 8F).
Widths of fibrils at their thinnest were approximately 2-3 nm. The
rHaPrP-res.sup.(spon) fibrils preferentially clustered in what
appeared to be doublets, with total widths of 6-8 nm, while those
of rHaPrP-res.sup.(Sc) formed larger side by side clusters of up to
36 nm in width. Bars designate 100 nm.
[0022] FIGS. 9A-9B are a series of graphs showing Fourier transform
infrared spectroscopy (FTIR) spectroscopy of rHaPrP-res.sup.(Sc)
and rHaPrP-res.sup.(spon). To compare the secondary structures of
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon), samples were
prepared using three sequential rounds of rPrP-PMCA so that the
original HaPrP.sup.Sc remaining in the seeded sample was
<0.0001% of the total prion protein analyzed. Portions of each
sample were left undigested (FIG. 9A) or digested with PK (FIG. 9B)
and analyzed by FTIR. The spectrum of the rHaPrP-sen substrate is
shown for comparison. Overlaid spectra are from independent
preparations. As expected, rHaPrP-sen had an absorbance maximum at
.about.1652 cm.sup.-1, consistent with prominent .alpha.-helical
and/or disordered secondary structures. In contrast, both
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon) displayed prominent
bands at lower wavenumbers (1615-1628 cm.sup.-1), indicating higher
proportions of .beta.-sheet. However, the location of the bands
differed between the two types of rHaPrP-res. Without PK treatment,
the rHaPrP-res.sup.(Sc) had maxima at 1628 and 1615 cm.sup.-1,
whereas rHaPrP-res.sup.(spon) peaked at 1625 cm.sup.-1. After PK
digestion of both types of rHaPrP-res, the intensities of bands in
the region associated with the .alpha.-helix and/or disordered
structures were attenuated. Prominent differences remained between
rHaPrP-res.sup.(Sc), with maxima at 1659, 1647, 1637 and 1628
cm.sup.-1, and rHaPrP-res.sup.(spon), with maxima at 1664 and 1627
cm.sup.-1. These spectral differences could be due to differences
in conformation, PK-resistant polypeptide chain length, or both.
Precise assignments of these bands are uncertain, but the 1664
cm.sup.-1 band is often associated with turns, and the 1659 and
1647 cm.sup.-1 bands with loops or helices, and disordered
structures, respectively. Of particular interest is the 1637
cm.sup.-1 band of PK-digested rHaPrP-res.sup.(Sc). This band also
features prominently in the spectrum of 263K HaPrP.sup.Sc (spectrum
of PK-treated sample is shown in FIG. 9B) and is absent from the
spectrum of the DY strains of HaPrP.sup.Sc, indicating that
strain-dependent structure associated with the 1637 cm.sup.-1 band
in 263K HaPrP.sup.Sc was replicated in rHaPrP-res.sup.(Sc). This
provides further evidence of the conformational fidelity of
rPrP-PMCA amplification.
[0023] FIGS. 10A-10B are a series of digital images of gels showing
that tube shaking supports ultra-sensitive prion-seeded conversions
of rPrP-sen. Purified PrPSc (FIG. 10A) or scrapie brain homogenate
(FIG. 10B) were used to seed the conversion of rHaPrP-sen to
protease-resistant forms in QUIC reactions performed in 0.1% sodium
dodecyl sulfate (SDS) and 0.1% TRITON.RTM. X-100
(C.sub.14H.sub.22O(C.sub.2H.sub.4O).sub.n, also known as
octylphenoxypolyethoxyethanol; Octoxynol-9; 4-octylphenol
polyethoxylate; or polyethylene glycol
p-(1,1,3,3-tetramethylbutyl)-phenyl ether, octyl phenol ethoxylate,
polyoxyethylene octyl phenyl ether), in PBS. PK digestions and
immunoblotting of reaction aliquots were performed as described in
Example 8. The C-terminal polyclonal antibody R20 was used in the
immunoblots. Circles designate the 17-kDa rHaPrP-res.sup.(Sc) band
and brackets designate the position of the .ltoreq.13 kDa
rHaPrP-res.sup.(Sc) bands. FIG. 10A shows a comparison of
PK-resistant QUIC reaction products from duplicate 24-hour unshaken
reactions and reactions shaken with or without 0.1 mm glass cell
disruption beads (Scientific Industries). 50 .mu.l reactions were
seeded containing 0.1 mg/ml (4 .mu.M) hamster rPrP-sen with 10 ng
of purified hamster PrPSc and subjected the tubes to cycles of 2
minutes of shaking and 28 minutes without shaking at 37.degree. C.
Enhanced rHaPrP-res.sup.(Sc) formation was noted in the shaken
reactions, but the beads were not influenced. 100 ng of rPrP-sen
without PK-treatment is shown in lane 1. FIG. 10B shows 20-hour
QUIC reactions performed with the designated rPrP-sen
concentrations, reaction volumes, and seed amounts. The seed
amounts indicate the estimated quantity of PrPSc added in 2-.mu.l
aliquots of scrapie brain homogenate diluted in 1% normal brain
homogenate. Lanes 6, 12, 18, and 24 received aliquots of only 1%
normal brain homogenate. The tubes were subjected to cycles of 10
seconds of shaking and 110 seconds without shaking. The asterisk
marks the position of rHaPrP-res.sup.(spon) bands.
[0024] FIG. 11 is a pair of digital images of gels showing that
extended reactions can enhance QUIC sensitivity to small amounts of
scrapie brain homogenate seed. QUIC reactions were performed with
0.1 mg/ml rPrP-sen and the designated reaction volumes and seed
amounts using the shaking cycle and buffer conditions described for
FIG. 10B. Two digital images are shown, 65-hour (upper blot) and
95-hour (lower blot) QUIC reactions were performed as using
100-.mu.l reaction volumes and dilutions of scrapie brain
homogenate containing the designated amount of PrP.sup.Sc. The
lanes marked `none` received comparable amounts of normal brain
homogenate only. Antiserum R20 was used for these blots. Open
circles designate the 17-kDa rHaPrP-res.sup.(Sc) band and brackets
designate the positions of the 10-13 kDa rHaPrP-res.sup.(Sc) or
rHaPrP-res.sup.(spon) bands. The positions of molecular mass
markers are designated in kDa on the left.
[0025] FIG. 12 is a digital image of a gel showing the results of
serial QUIC reactions. For the first round, QUIC reactions were
performed under the conditions described in the brief description
of FIG. 11B, except for the use of 48-hour reaction times and
reduced detergent concentrations (0.05 SDS and 0.05% TRITON.RTM.
X-100). For the second round, 10% of the volume of the first round
reaction products were diluted into 9 volumes of reaction buffer
containing fresh rPrP-sen. PK-digested products were immunoblotted
using D13 primary antibody. Open circles designate the 17-kDa
rHaPrP-res.sup.(Sc) band. The positions of molecular mass markers
are designated in kDa on the left.
[0026] FIGS. 13A-13B are digital images of gels showing the results
of seeding QUIC reactions with CSF. Aliquots (2 .mu.l) of CSF taken
from normal hamsters (n=3) or hamsters in the clinical phase of
scrapie (n=6) were used to seed QUIC reactions using the conditions
described for FIG. 12. Immunoblots of the PK-digested products of
the first 48-hour round are shown in FIG. 13A. Ten percent of each
first round reaction volume was used to seed a second 48-hour round
of QUIC and the PK-digested products of the latter are shown in
FIG. 13B. Antibodies R20 (top) and D13 (bottom) were used for the
immunoblots. Lane 1 of each panel shows 100 ng HaPrP-sen without PK
treatment. The positions of the 17-kDa) rHaPrP-res.sup.(Sc) band
are marked with a circle. The positions of molecular mass markers
are designated in kDa on the left. These 37.degree. C. reactions
contained 0.05% SDS and 0.05% TRITON.RTM. X-100 in PBS and were
shaken at 1500 rpm for 10 seconds every 2 minutes. FIG. 13A shows
immunoblots with antibody R20 of the PK-digested products of the
first 48-h round. FIG. 13B is an R20 immunoblot showing products of
second-round reactions seeded with 10% of each first round reaction
volume.
[0027] FIGS. 14A-14B are digital images of gels showing
ultrasensitive prion-seeded conversions of rPrP-sen in single-round
46-hour QUIC reactions at 45.degree. C. Scrapie brain homogenate
was used to seed the conversion of rHaPrP-sen to protease-resistant
forms in QUIC reactions (0.1% SDS and 0.1% TRITON.RTM. X-100, in
PBS). The reactions were shaken at 1500 rpm for 10 s every 2 min.
PK digestions and immunoblotting of reaction aliquots were
performed with the C-terminal antibody R20. Circles designate the
17-kDa rHaPrP-res (Sc) band and brackets designate the position of
the .ltoreq.13 kDa rHaPrP-res(Sc) bands. FIG. 14A illustrates the
sensitivity of the reaction with dilutions of normal brain
homogenate (NBH) and scrapie brain homogenate (ScBH) as seeds. The
ScBH seeds contained the designated amounts of PrPSc. The NBH was
0.00001% w/v in the reaction, which is equivalent to that of the
ScBH seed dilution containing 1 pg of PrPSc. The NBH and ScBH
samples were diluted to the designated levels in 1% N-2 supplement
(Invitrogen), except in the lanes marked 1 pg*, which were diluted
in 0.1% N-2. Either NBH or N-2 can be used as a diluent. FIG. 14B
is an analysis of multiple negative controls under the reactions
conditions of FIG. 14A. The ScBH seeds contained 1 pg of PrPSc
while the NBH content in the negative controls are as designated.
The lanes marked none were seeded with the diluent for the brain
homogenates, i.e., N-2. Molecular mass markers are designated on
the left.
[0028] FIGS. 15A-15B are a pair of digital images of gels showing
that extended reactions can enhance QUIC sensitivity to small
amounts of scrapie brain homogenate seed. In FIG. 15A, 40-hour QUIC
reactions were performed with 0.1 mg/ml rPrP-sen and the designated
reaction volumes and seed amounts using the shaking cycle and
buffer conditions described for FIG. 10B. The upper and lower
panels show immunoblots performed using antibody R20 and D13,
respectively (PrP epitope residues shown in parentheses). In FIG.
15B, 65-hour (upper blot) and 95-hour (lower blot) QUIC reactions
were performed as in FIG. 15A using 100-.mu.l reaction volumes and
dilutions of scrapie brain homogenate containing the designated
amount of PrP.sup.Sc. The lanes marked `none` received comparable
amounts of normal brain homogenate only. Antiserum R20 was used for
these blots. Open circles designate the 17-kDa rHaPrP-res.sup.(Sc)
band and brackets designate the positions of the 10-13 kDa
rHaPrP-res.sup.(Sc) or rHaPrP-res.sup.(spon) bands. The positions
of molecular mass markers are designated in kDa on the left. FIG.
11 and FIG. 15B provide results from the same experiment.
[0029] FIGS. 16A-16D are a series of digital images showing the
effect of temperature on QUIC reaction products and kinetics. QUIC
reactions were seeded at different temperatures and reaction times
with scrapie brain homogenates (diluted in N2) containing the
designated amount of PrP.sup.Sc or normal brain homogenate (NBH) at
the dilution used for the 100-fg scrapie brain homogenate sample.
The PK-digested products were immunoblotted with antibody R20. Rows
FIGS. 16A, 16B, 16C, and 16D show reactions performed at 37.degree.
C., 45.degree. C., 55.degree. C. and 65.degree. C., respectively.
Successive columns of blots show reactions run for 4, 8 and 18
hours. All of the QUIC reactions were run in 0.1% SDS and 0.1%
TRITON.RTM. X-100 in PBS with 0.1 mg/ml rPrP-sen with 60 seconds of
shaking at 1500 rpm and 60 seconds of rest. The reaction products
were digested with PK under the Sarkosyl-containing conditions
described in Example 8. The positions of molecular mass markers are
designated in kDa on the left in the first column or by
corresponding tick marks by the other columns. The open circles
designate the position of the 17 kDa band and the bracket the 10-13
kDa bands.
[0030] FIGS. 17A-17B illustrate the effect of shaking variations on
the QUIC reaction. QUIC reactions were subjected to cycles of 10
seconds shaking and 110 seconds res (top panel, FIG. 17A) with
reactions shaken for 60 seconds and rested for 60 seconds (bottom
panel, FIG. 17B). These reactions were seeded with scrapie brain
homogenate (NBH) at dilutions identical to that used for the 10 fg
scrapie brain homogenate sample. The reaction temperature was
45.degree. C. and the QUIC buffer conditions, PK-digestion and
immunoblot protocols were as described for FIG. 18. The positions
of molecular mass markers are designated in kDa on the left; the
open circles designate the position of the 17 kDa band and the
bracket the 10-13 kDa bands.
[0031] FIG. 18 illustrates the effect of detergent conditions on PK
digestion of QUIC reaction products. QUIC reactions performed at
45.degree. C. were seeded with scrapie brain homogenates (diluted
in N2) containing 100 fg of PrP.sup.Sc or the same dilution of
normal brain homogenate (NBH). The shaking cycle was 10 seconds on
and 110 second off, and the buffer conditions were as described in
connection with FIG. 16. 10 .mu.l aliquots of the reaction products
were mixed with 4 .mu.l of the designated detergent solutions and
digested with 7 .mu.g/ml PK (final concentration) for 60 minutes at
37.degree. C. The samples were then immunoblotted using R20
antibody. The positions of molecular mass markers are designated in
kDa on the left; the open circles designate the position of the 17
kDa band and the bracket the 10-13 kDa bands. The upper band
represents residual full length rPrP molecules.
[0032] FIG. 19 is a digital image of a blot showing the sensitivity
of an 18-hour QUIC reaction at 55.degree. C. QUIC reactions were
seeded with scrapie brain homogenates (diluted in N2) containing
the designated amount of PrP.sup.Sc or normal brain homogenate
(NBH) at the dilution used for the 10-fg scrapie brain homogenate
sample. Reaction buffer constituents, PK-digestion conditions, and
immunoblotting were as described in the legend to FIG. 12. The
positions of molecular mass markers are designated in kDa on the
left. The open circles designate the position of the 17 kDa band
and the bracket the 10-13 kDa bands.
[0033] FIG. 20 shows blots from a QUIC reaction seeded either with
dilutions of brain homogenate from a variant CJD patient containing
100 fg, 10 fg, or 1 fg of PrP-res or, as a negative control, a
dilution of a non-CJD human brain homogenate (from an Alzheimer's
disease patient) equivalent to the 100-fg vCJD brain homogenate
dilution. The recombinant PrP substrate in these reactions was the
Syrian hamster PrP sequence (residues 23-231). This was a
single-round reaction at 50.degree. C. for either 8 hours (top
blots) or 18 hours (bottom blots). The primary antibody used to
detect the rPrP-res[CJD] reaction products was monoclonal Ab 3F4,
which has an epitope within residues 106-112, and thus, is only
expected to detect the 17-kDa rPrP-res[CJD] product and not the
smaller bands that are detected by more C-terminally reactive
antibodies. Six separate reactions were performed with each type or
dilution of seed and the number of rPrP-res[CJD]-positive reactions
per 6 replicates is indicated below each set of replicates on the
blots.
SEQUENCE LISTING
[0034] The Sequence Listing is submitted as an ASCII text file
4239-77856-17_Sequence_Listing.txt, Jul. 31, 2017, .about.21.8 KB],
which is incorporated by reference herein.
[0035] The nucleic acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of
each nucleic acid sequence is shown, but the complementary strand
is understood as included by any reference to the displayed strand.
In the accompanying sequence listing:
[0036] SEQ ID NO: 1 is an amino acid sequence of a recombinant
Syrian golden hamster proteinase K-sensitive prion protein.
TABLE-US-00001 kkrpkpgg wntggsrypg qgspggnryp pqgggtwgqp hgggwgqphg
ggwgqphggg wgqphgggwg qgggthnqwn kpnkpktsmk hmagaaaaga vvgglggyml
gsamsrpmlh fgndwedryy renmnrypnq vyyrpvdqyn nqnnfvhdcv nitikqhtvt
tttkgenfte tdvkmmervv eqmcvtqyqk esqayydgrr s
[0037] SEQ ID NO: 2 is an amino acid sequence of a recombinant
mouse (Prnp-a) proteinase K-sensitive prion protein.
kkrpkpgg wntggsrypg qgspggnryp pqggtwgqph gggwgqphgg swgqphggsw
gqphgggwgq gggthnqwnk pskpktnlkh vagaaaagav vgglggymlg samsrpmihf
gndwedryyr enmyrypnqv yyrpvdqysn qnnfvhdcvn itikqhtvtt ttkgenftet
dvkmmervve qmcvtqyqke sqayydgrrs
[0038] SEQ ID NO: 3 is an amino acid sequence of a recombinant
human (129M) proteinase K-sensitive prion protein.
kkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg
wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyml gsamsrpiih
fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte
tdvkmmervv eqmcitqyer esqayyqrgs s
[0039] SEQ ID NO: 4 is an amino acid sequence of a recombinant
human (129V) proteinase K-sensitive prion protein.
kkrpkpgg wntggsrypg qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg
wgqphgggwg qgggthsqwn kpskpktnmk hmagaaaaga vvgglggyvl gsamsrpiih
fgsdyedryy renmhrypnq vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte
tdvkmmervv eqmcitqyer esqayyqrgs s
[0040] SEQ ID NO: 5 is an amino acid sequence of a recombinant
bovine (6-octarepeat) proteinase K-sensitive prion protein.
kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq phgggwgqph
gggwgqphgg gwgqphgggg wgqggthgqw nkpskpktnm khvagaaaag avvgglggym
lgsamsrpli hfgsdyedry yrenmhrypn qvyyrpvdqy snqnnfvhdc vnitvkehtv
ttttkgenft etdikmmery veqmcitqyq resqayyqrgas
[0041] SEQ ID NO: 6 is an amino acid sequence of a recombinant
ovine (136A 154R 171Q) proteinase K-sensitive prion protein.
kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq phgggwgqph
gggwgqphgg ggwgqggshs qwnkpskpkt nmkhvagaaa agavvgglgg ymlgsamsrp
lihfgndyed ryyrenmyry pnqvyyrpvd qysnqnnfvh dcvnitvkqh tvttttkgen
ftetdikime rvveqmcitq yqresqayyq rga
[0042] SEQ ID NO: 7 is an amino acid sequence of a recombinant Deer
(96G 132M 138S) proteinase K-sensitive prion protein.
TABLE-US-00002 kkrpkp gggwntggsr ypgqgspggn ryppqggggw gqphgggwgq
phgggwgqph gggwgqphgg ggwgqggths qwnkpskpkt nmkhvagaaa agavvgglgg
ymlgsamsrp lihfgndyed ryyrenmyry pnqvyyrpvd qynnqntfvh dcvnitvkqh
tvttttkgen ftetdikmme rvveqmcitq yqresqayyq rgas
[0043] SEQ ID NO: 8 is an amino acid sequence of a full-length
Syrian golden hamster proteinase K-sensitive prion protein.
TABLE-US-00003 mwtdvglckk rpkpggwntg gsrypgqgsp ggnryppqgg
gtwgqphggg wgqphgggwg qphgggwgqp hgggwgqggg thnqwnkpsk pktnmkhmag
aaaagavvgg lggymlgsam srpmmhfgnd wedryyrenm nrypnqvyyr pvdqynnqnn
fvhdcvniti kqhtvttttk genftetdik imervveqmc ttqyqkesqa yydgrrssav
lfssppvill isfliflmvg
[0044] SEQ ID NO: 9 is an amino acid sequence of a full-length
mouse (Prnp-a) proteinase K-sensitive prion protein.
TABLE-US-00004 manlgywlla lfvtmwtdvg lckkrpkpgg wntggsrypg
qgspggnryp pqggtwgqph gggwgqphgg swgqphggsw gqphgggwgq gggthnqwnk
pskpktnlkh vagaaaagav vgglggymlg samsrpmihf gndwedryyr enmyrypnqv
yyrpvdqysn qnnfvhdcvn itikqhtvtt ttkgenftet dvkmmervve qmcvtqyqke
sqayydgrrs sstvlfsspp villisflif livg
[0045] SEQ ID NO: 10 is an amino acid sequence of a full-length
human (129M) proteinase K-sensitive prion protein.
TABLE-US-00005 manlgcwmlv lfvatwsdlg lcldcrpkpgg wntggsrypg
qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn
kpskpktnmk hmagaaaaga vvgglggyml gsamsrpiih fgsdyedryy renmhrypnq
vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer
esqayyqrgs smvlfssppv illisflifl ivg
[0046] SEQ ID NO: 11 is an amino acid sequence of a full-length
human (129V) proteinase K-sensitive prion protein.
TABLE-US-00006 manlgcwmlv lfvatwsdlg lcldcrpkpgg wntggsrypg
qgspggnryp pqggggwgqp hgggwgqphg ggwgqphggg wgqphgggwg qgggthsqwn
kpskpktnmk hmagaaaaga vvgglggyvl gsamsrpiih fgsdyedryy renmhrypnq
vyyrpmdeys nqnnfvhdcv nitikqhtvt tttkgenfte tdvkmmervv eqmcitqyer
esqayyqrgs smvlfssppv illisflifl ivg
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
I. Overview of Several Embodiments
[0047] Disclosed herein is an ultrasensitive method, termed
rPrp-res amplification, for detecting PrP.sup.Sc that allows the
use of purified recombinant rPrP-sen as a substrate for seeded
polymerization. The resulting assay is much faster than previous
PMCA methods, and the use of rPrP-sen facilitates improved prion
assays and fundamental studies of structure and formation of
PrP.sup.Sc. These methods can be used to diagnose a variety of
diseases in animal and human subjects, and reduce the time
necessary for high sensitivity detection of PrP-res in samples.
Thus, the present disclosure also enables high throughput, accurate
and sensitive screening of samples, as well as diagnosis of
clinical disease.
[0048] In certain embodiments, the methods are used to diagnose a
prion disease or a disease induced by a protein conformation
change, such as a conformational change in PrP-sen. The disease can
be a transmissible spongiform encephalopathy, such as bovine
spongiform encephalopathy (BSE) in a cow, whereas in sheep, the
methods are used to diagnose scrapie, and in deer, elk, and moose
the methods are used to diagnose CWD. The method also enables the
rapid testing of live animals for infection to protect against
unnecessary culling of herds or inadvertent introduction of prions
into the food chain.
[0049] The disclosed methods also are used to diagnose humans and
human diseases. Prion diseases that the methods detect in humans
include but are not limited to Creutzfeldt-Jakob disease (CJD),
kuru, fatal familial insomnia, Gerstmann-Straussler-Scheinker
disease, and sporadic fatal insomnia. As when used for the
diagnosis of animal diseases, the disclosed methods offer
significant advantages over available methods for diagnosis of
these neurologic disorders. For instance, cognitive tests and
clinical signs currently used for diagnosis of CJD can only
indicate a probable diagnosis, and conventional PMCA takes up to
three weeks to perform, whereas the disclosed methods provide an
objective method by which positive diagnosis can be made within 1-2
days with little chance of false positive or false negative
results. Additionally, the sensitivity of the test enables the
detection of disease from peripheral tissues, such as blood and
cerebral spinal fluid (CSF), which is much less invasive and
expensive than brain biopsy procedures. The methods also provide
sensitivity that is sufficiently high to detect or diagnose disease
prior to the onset of clinical symptoms.
[0050] One embodiment of the disclosure is a method for detecting
PrP-res in a sample. The method includes (a) mixing the sample with
purified rPrP-sen to make a reaction mix (b) performing a primary
reaction that includes (i) incubating the reaction mix to permit
the coaggregation of the rPrP-sen with the PrP-res that may be
present in the reaction mix; (ii) agitating any aggregates formed
during step (i); and (iii) repeating steps (i) and (ii) one or more
times. In step (i) of the primary reaction, aggregation of the
rPrP-sen with the PrP-res results in a conversion of the rPrP-sen
to rPrP-res.sup.(Sc). These amplification steps are then followed
by (c) detecting) rPrP-res.sup.(Sc) in the reaction mix, wherein
detection of rPrP-res.sup.(Sc) in the reaction mix indicates that
PrP-res was present in the sample. In some examples, steps (b)(i)
and (b)(ii) are repeated from about 1 to about 200 times. In other
examples, serial amplification is performed by removing a portion
of the reaction mix and incubating it with additional rPrP-sen.
[0051] The reaction can be carried out by maintaining incubation
conditions to inhibit production of rPrP-res.sup.(spon) which in
the past has competed with the desired reaction and may have
contributed to the conclusion that cyclic amplification could not
be carried out with rPrP-sen. The detailed description describes a
number of ways to inhibit production of rPrP-res.sup.(spon), for
example by one or more of (a) agitating the aggregates by shaking
the reaction mix without sonication; (b) incubating the reaction
mix in 0.05% to 0.1% of a detergent; (c) incubating the reaction
mix at 37.degree. C.-60.degree. C.; or (d) incubating the reaction
mix for no more than 2, 4, 6, 8, 16 or 20 hours at higher reaction
temperatures. In certain examples, any combination of (a)-(d) or
all of them are used to inhibit rPrP-res.sup.(spon) production,
such that the amount of rPrP-res.sup.(spon) is less than 20% (or
even less than 15% or 10%) of that of rPrP-res.sup.(Sc) generated
(in reactions seeded with samples containing PrP-res). The
detergent may be a mixture of detergents, such as a mixture of an
anionic and nonionic detergent, such as SDS and TRITON.RTM.
X-100.
[0052] In certain embodiments, the method also includes the step of
performing a serial amplification reaction before detecting
rPrP-res.sup.(Sc) in the reaction mix, wherein performing the
serial reaction includes removing a portion of the reaction mix and
incubating it with additional rPrP-sen. In other embodiments,
detecting the PrP-res includes detecting rPrP-res.sup.(Sc)
aggregates in the reaction mix. Still other embodiments also
include digesting the reaction mix with proteinase K prior to
detecting rPrP-res.sup.(Sc) in the reaction mix. In certain
examples, detecting)rPrP-res.sup.(Sc) includes using an antibody
that specifically binds to prion protein, for instance D13, R18, or
R20 antibodies.
[0053] In some embodiments of the disclosure, the PrP-res includes
mammalian prion protein, and in certain examples, the rPrP-sen
includes a detectable label. In other embodiments, incubating the
reaction mix includes incubating the reaction mix at about 25 to
70.degree. C., and in particular examples incubating the reaction
mix includes incubating the reaction mix at about 37 to 55.degree.
C., or 45 to 55.degree. C. In some examples, incubating the
reaction mix includes incubating the reaction mix between
agitations for about 1 to about 180 minutes, and in particular
examples incubating the reaction mix includes incubating the
reaction mix for 1 min or for about 60 to about 120 minutes, such
as about 60, about 70, about 80, about 90, about 100, about 110 or
about 110 minutes, for example about 70 to about 100 minutes. In
other examples, the reaction mix can be incubated for about 1,
about 2, about 5, about 10, about 20, about 30, about 40 minutes
between agitations. The total reaction time, including agitation
and incubation can be about 2 to about 48 hours, such as about 4,
about 6, about 8, about 16, about 20, about 24, about 36, about 42,
or about 48 hours.
[0054] In some examples of rPrP-PMCA, agitating the aggregates
includes sonicating the reaction mix, and in some examples,
agitating the reaction mix includes sonicating the reaction mix for
about 1-120 seconds, or in other examples, for about 40 seconds. In
other embodiments, termed QUIC, agitating the reaction mix includes
shaking the reaction mix for about 1-120 seconds, for instance for
about 10 or 60 seconds. The primary reaction includes, in some
embodiments, (a) incubating the reaction mix for approximately 60
minutes; and (b) sonicating the reaction mix for approximately 40
seconds. In some examples, steps (a) and (b) are repeated for
approximately 1-48 hours.
In other examples, the reaction mixture further includes an ionic
(such as an anionic) and a nonionic detergent, such as SDS and
TRITON.RTM. (TX)-100, for example, from about 0.05% to about 0.1%
SDS and from about 0.05% to about 0.1% TX-100. Other suitable
non-ionic detergents include Bis(polyethylene glycol bis[imidazoyl
carbonyl]), Decaethylene glycol monododecyl ether,
N-Decanoyl-N-methylglucamine, n-Decyl a-D-glucopyranoside, Decyl
b-D-maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl
a-D-maltoside, n-Dodecyl b-D-maltoside, n-Dodecyl b-D-maltoside,
SigmaUltra, Heptaethylene glycol monodecyl ether, Heptaethylene
glycol monododecyl ether, Heptaethylene glycol monotetradecyl
ether, n-Hexadecyl b-D-maltoside, Hexaethylene glycol monododecyl
ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol
monooctadecyl ether, Hexaethylene glycol monotetradecyl ether,
Methyl-6-O--(N-heptylcarbamoyl)-a-D-glucopyranoside, Nonaethylene
glycol monododecyl ether, N-Nonanoyl-N-methylglucamine,
N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,
Octaethylene glycol monododecyl ether, Octaethylene glycol
monohexadecyl ether, Octaethylene glycol monooctadecyl ether,
Octaethylene glycol monotetradecyl ether,
Octyl-b-D-glucopyranoside, Pentaethylene glycol monodecyl ether,
Pentaethylene glycol monododecyl ether, Pentaethylene glycol
monohexadecyl ether, Pentaethylene glycol monohexyl ether,
Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol
monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene
glycol ether W-1, Polyoxyethylene 10 tridecyl ether,
Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl
ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate,
Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate,
Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25
propylene glycol stearate, Saponin from Quillaj a bark, SPAN.RTM.
(Nos. 20, 40, 60, 65 80, or 85), Tergitol (Type 15-S-12, Type
15-S-30, Type 15-S-5, Type 15-S-7, Type 15-S-9, Type NP-10, Type
NP-4, Type NP-40, Type NP-7, Type NP-9, MIN FOAM 1.times., MIN FOAM
2.times., Type TMN-10, Type TMN-6), Tetradecyl-b-D-maltoside,
Tetraethylene glycol monodecyl ether, Tetraethylene glycol
monododecyl ether, Tetraethylene glycol monotetradecyl ether,
Triethylene glycol monodecyl ether, Triethylene glycol monododecyl
ether, Triethylene glycol monohexadecyl ether, Triethylene glycol
monooctyl ether, Triethylene glycol monotetradecyl ether,
TRITON.RTM..RTM. CF-21, TRITON.RTM. CF-32, TRITON.RTM. DF-12,
TRITON.RTM. DF-16, TRITON.RTM. GR-5M, TRITON.RTM. X-100,
TRITON.RTM. X-102, TRITON.RTM. X-15, TRITON.RTM. X-151, TRITON.RTM.
X-207, TRITON.RTM. X-100, TRITON.RTM. X-114, TRITON.RTM. X-165,
TRITON.RTM. X-305, TRITON.RTM. X-405, TRITON.RTM. X-45, TRITON.RTM.
X-705-70, TWEEN.RTM. 20, TWEEN.RTM. 21, TWEEN.RTM. 40, TWEEN.RTM.
60, TWEEN.RTM. 6, TWEEN.RTM. 65, TWEEN.RTM. 80, TWEEN.RTM. 81,
TWEEN.RTM. 85, Tyloxapol, and n-Undecyl b-D-glucopyranoside. Other
suitable anionic detergents of use include Chenodeoxycholic acid,
Chenodeoxycholic acid sodium salt, Cholic acid, ox or sheep bile,
Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid methyl
ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide,
Docusate sodium salt, Glycochenodeoxycholic acid sodium salt,
Glycocholic acid hydrate (Glycodeoxycholic acid monohydrate,
Glycodeoxycholic acid sodium salt, Glycolithocholic acid 3-sulfate
disodium salt, Glycolithocholic acid ethyl ester),
N-Lauroylsarcosine, Lithium dodecyl sulfate, Niaproof 4,
TRITON.RTM. QS-15, TRITON.RTM. QS-44, 1-Octanesulfonic acid sodium
salt, Sodium 1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-dodecanesulfonate, Sodium 1-heptanesulfonate anhydrous, Sodium
1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate, Sodium
2-bromoethanesulfonate, Sodium cholate hydrate, Sodium choleate,
Sodium deoxycholate, Sodium deoxycholate monohydrate, Sodium
hexanesulfonate, Sodium octyl sulfate, Sodium pentanesulfonate,
Sodium taurocholate, Taurochenodeoxycholic acid sodium salt,
Taurodeoxycholic acid sodium salt monohydrate, Taurodeoxycholic
acid sodium salt monohydrate, Taurohyodeoxycholic acid sodium salt
hydrate, Taurolithocholic acid 3-sulfate disodium salt
Tauroursodeoxycholic acid sodium salt, TRITON.RTM. X-200M
TRITON.RTM. XQS-20, TRIZMA.RTM. dodecyl sulfate, and
Ursodeoxycholic acid. The anionic and ionicdetergents can be used
for example, at a concentration of 0.01% to 0.5%, such as 0.05% to
0.1%.
[0055] In some embodiments, the source of the recombinant rPrP-sen
is the same species as the source of the sample or it is of a
different species as the source of the sample. It has particularly
been found that rHaPrP-sen is well suited to the amplification
reaction, and can be used to amplify target protein in a target
from species other than hamster. Some examples of the method
include a rPrP-sen that is bovine, ovine, hamster, rat, mouse,
canine, feline, cervid, human, or non-human primate rPrP-sen. In
certain examples, the rPrP-sen includes amino acids 23-231 (SEQ ID
NO: 1) of Syrian golden hamster prion protein (SEQ ID NO: 8), amino
acids 23-231 (SEQ ID NO: 2) of mouse prion protein (SEQ ID NO: 9),
amino acids 23-231 (SEQ ID NO: 3) of human (129M) prion protein
(SEQ ID NO: 10), amino acids 23-231 (SEQ ID NO: 4) of human (129V)
prion protein (SEQ ID NO: 11), amino acids 25-241 (SEQ ID NO: 5) of
bovine (6-octarepeat) prion protein, amino acids 25-233 (SEQ ID NO:
6) of ovine (136A 154R 171Q) prion protein, or amino acids 25-234
(SEQ ID NO: 7) of deer (96G 132M 138S) prion protein. However,
fragments of rPrP-sen can also be used, such as but not limited to
a fragment comprising amino acids 23-231 of rPrP-sen. Fragments
include amino acids 30-231, amino acids 40-231, amino acids 50-231,
amino acids 60-231, amino acids 70-231, amino acids 80-231 or amino
acids 90-231 of mouse, human, hamster, bovine, ovine or deer prion
protein. A functional fragment of rPrP-sen can aggregate with
PrP-res and result in a conversion of the rPrP-sen to
rPrP-res.sup.(Sc). It should be noted that chimeric rPrP-sen,
wherein a portion of the protein is from one species, and a portion
of the protein is from another species, can also be utilized. In
one example about 10 to about 90%, such as about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70% about 80% or
about 90% of the rPrP-sen is from one species, and,
correspondingly, about 90%, about 80%, about 70%, about 60%, about
50%, about 40%, about 30%, about 20% or about 10% is from another
species. Chimeric proteins can include, for example, hamster
rPrP-sen and rPrP-sen from another species, such as human
PrP-sen.
[0056] Particular examples include a sample that is a tissue sample
from an animal, for instance a brain sample, a peripheral organ
sample, feces, urine, mucosal secretions, or a CSF sample. In even
more particular examples, the peripheral organ sample includes
blood, tonsil, nasal tissue, spleen, or another lymphoid organ.
[0057] Detecting PrP-res.sup.(Sc) in the reaction mix includes, in
some embodiments, performing a Western blot, an ELISA assay, a CDI
assay, a DELPHIA assay, a strip immuno-chromatographic assay, a
spectroscopic assay, a fluorescence assay, or a radiometric assay.
In certain examples, the ELISA assay is a two-site immunometric
sandwich ELISA. In particular examples, the prion can be detected
in a sample containing at least 1000 PrP.sup.Sc molecules. In still
more particular examples, the method further includes inactivating
residual PrP-res in the reaction mix. In yet other examples, the
method is a method of diagnosing a prion disease.
[0058] Also disclosed herein are kits for detection of a prion in a
sample that include rPrP-sen and at a reaction mix buffer. In some
embodiments, the reaction mix buffer includes SDS and TX-100, and
in other embodiments, the rPrP-sen is lyophilized. In certain
examples, the kit also includes one or more of (a) a
decontamination solution; (b) a positive control; (c) a negative
control; or (d) reagents for the detection of rPrP-res.sup.(Sc). In
particular examples, the reagents for detection of
rPrP-res.sup.(Sc) include antibodies.
[0059] In yet other embodiments, a rPrP-res amplification method is
disclosed for detecting PrP-res in a sample by
[0060] (a) mixing the sample with purified rPrP-sen to make a
reaction mix; and performing an amplification reaction in a single
round without serial amplification, by incubating the reaction
mixture to permit coaggregation of the rPrP-sen with the PrP-res
that may be present in the reaction mix, wherein coaggregation of
the rPrP-sen with the PrP-res results in a conversion of the
rPrP-sen to the rPrP-res.sup.(Sc). Using this method it is possible
to sensitively detect PrP-res in a sample under a variety of
conditions, such as conditions (b) through (e) below:
[0061] (b) detecting PrP-res in a sample containing as little as
100 ag PrP-res by incubating the reaction mixture at 45.degree. C.
for about 46 hours and intermittently shaking the reaction mixture
without sonication, and rPrP-res.sup.(Sc) is detected as an
indicator of the initial presence of PrP-res;
[0062] (c) detecting PrP-res in a sample containing as little as 1
fg PrP-res by incubating the reaction mixture at 55.degree. C. for
about 18 hours and intermittently shaking the reaction mixture
without sonication, and rPrP-res.sup.(Sc) is detected as an
indicator of the initial presence of PrP-res;
[0063] (d) detecting PrP-res in a sample containing as little as 10
fg PrP-res by incubating the reaction mixture at 55.degree. C. for
about 8 hours and intermittently shaking the reaction mixture
without sonication, and rPrP-res.sup.(Sc) is detected as an
indicator of the initial presence of PrP-res; and
[0064] (e) detecting PrP-res in a sample containing as little as
100 fg PrP-res by incubating the reaction mixture at 65.degree. C.
for about 4 hours and intermittently shaking the reaction mixture
without sonication, and rPrP-res.sup.(Sc) is detected as an
indicator of the initial presence of PrP-res.
II. Abbreviations
[0065] BH: brain homogenate
[0066] BSE: bovine spongiform encephalopathy
[0067] CJD: Creutzfeldt-Jakob disease
[0068] CSF: cerebral spinal fluid
[0069] CWD: chronic wasting disease
[0070] EEG: electroencephalogram
[0071] ELISA: enzyme linked immunosorbent assays
[0072] EUE: exotic ungulate encephalopathy
[0073] fCJD: familial Creutzfeldt-Jakob disease
[0074] FFI: fatal familial insomnia
[0075] GFP: green fluorescent protein
[0076] GSS: Gerstmann-Strassler Sheinker syndrome
[0077] GST: Glutathione S-transferase
[0078] HaPrP-res: hamster proteinase K resistant prion protein
[0079] HaPrP.sup.Sc: hamster proteinase K resistant prion
protein
[0080] HaPrP-sen: hamster proteinase K sensitive prion protein
[0081] iCJD: iatrogenic Creutzfeldt-Jakob disease
[0082] MBP: Maltose binding protein
[0083] NBH: normal brain homogenate
[0084] PBS: phosphate buffered saline
[0085] PK: proteinase K
[0086] PMCA: protein misfolding cyclic amplification
[0087] PrP-res: proteinase K resistant prion protein
[0088] PrP.sup.Sc: proteinase K resistant prion protein
[0089] PrP-sen: proteinase K sensitive prion protein
[0090] QUIC: quaking-induced conversion
[0091] RIA: radioimmunoassay
[0092] rHaPrP-res.sup.(vCJD): recombinant hamster proteinase K
resistant prion protein, variant Creutzfeldt-Jakob disease that
arises from seeding hamster PrP-res into a human sample
[0093] rPrP-res: recombinant proteinase K resistant prion
protein
[0094] rPrP-res.sup.(Sc): recombinant proteinase K resistant prion
protein seeded by
[0095] rPrP-res.sup.(spon): recombinant proteinase K resistant
prion protein that spontaneously arises without seeding (unseeded)
by rPrP-res
[0096] rPrP-sen: recombinant proteinase K sensitive prion protein
ScBH Scrapie brain homogenate
[0097] sCJD: sporadic Creutzfeldt-Jakob disease
[0098] SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel
electrophoresis
[0099] sFI: sporadic fatal insomnia
[0100] TSE: transmissible spongiform encephalopathy
[0101] TX-100: TRITON.RTM. X-100
[0102] vCJD: variant Creutzfeldt-Jakob disease
III. Terms
[0103] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular biology can be found in
Benjamin Lewin, Genes V, published by Oxford University Press, 1994
(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8).
[0104] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. The term "plurality" refers to two or
more. It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of this disclosure, suitable methods and materials are
described herein. The term "comprises" means "includes."
[0105] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0106] Aggregate: as used herein, includes aggregates, dimers,
multimers, and polymers of prion proteins, for instance aggregates,
dimers, multimers, and polymers of PrP-res, rPrP-res, or
rPrP-res.sup.(Sc).
[0107] Agitation: includes introducing any type of turbulence or
motion into a mixture or reaction mix, for examples by sonication,
stirring, or shaking. In some embodiments, agitation includes the
use of force sufficient to fragment rPrP-res.sup.(Sc) aggregates,
which disperses rPrP-res.sup.(Sc) aggregates and/or polymers to
facilitate further amplification. In some examples fragmentation
includes complete fragmentation, whereas in other examples,
fragmentation is only partial, for instance, a population of
aggregates can be about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 100% fragmented by agitation. Exemplary agitation
methods are described in the Examples section below.
[0108] Conservative variant: in the context of a prion protein,
refers to a peptide or amino acid sequence that deviates from
another amino acid sequence only in the substitution of one or
several amino acids for amino acids having similar biochemical
properties (so-called conservative substitutions). Conservative
amino acid substitutions are likely to have minimal impact on the
activity of the resultant protein. Further information about
conservative substitutions can be found, for instance, in Ben
Bassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et al.
(Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci.,
3:240-247, 1994), Hochuli et al. (Bio/Technology, 6:1321-1325,
1988) and in widely used textbooks of genetics and molecular
biology. In some examples, prion protein variants can have no more
than 1, 2, 3, 4, 5, 10, 15, 30, 45, or more conservative amino acid
changes. Conservative variants are discussed in greater detail in
section IV F of the Detailed Description.
[0109] In one example, a conservative variant prion protein is one
that functionally performs substantially like a similar base
component, for instance, a prion protein having variations in the
sequence as compared to a reference prion protein. For example, a
prion protein or a conservative variant of that prion protein, will
aggregate with rPrP-sen (or PrP.sup.Sc), for instance, and will
convert rPrP-sen to rPrP-res (or will be converted to rPrP-res). In
this example, the prion protein and the conservative variant prion
protein do not have the same amino acid sequences. The conservative
variant can have, for instance, one variation, two variations,
three variations, four variations, or five or more variations in
sequence, as long as the conservative variant is still
complementary to the corresponding prion protein.
[0110] In some embodiments, a conservative variant prion protein
includes one or more conservative amino acid substitutions compared
to the prion protein from which it was derived, and yet retains
prion protein biological activity. For example, a conservative
variant prion protein can retain at least 10% of the biological
activity of the parent prion protein molecule from which it was
derived, or alternatively, at least 20%, at least 30%, or at least
40%. In some preferred embodiments, a conservative variant prion
protein retains at least 50% of the biological activity of the
parent prion protein molecule from which it was derived. The
conservative amino acid substitutions of a conservative variant
prion protein can occur in any domain of the prion protein.
[0111] Disaggregate: To partially or complete disrupt an aggregate,
such as an aggregate of PrP-res, rPrP-res, or
rPrP-res.sup.(Sc).
[0112] Encode: any process whereby the information in a polymeric
macromolecule or sequence is used to direct the production of a
second molecule or sequence that is different from the first
molecule or sequence. As used herein, the term is construed
broadly, and can have a variety of applications. In some aspects,
the term "encode" describes the process of semi-conservative DNA
replication, wherein one strand of a double-stranded DNA molecule
is used as a template to encode a newly synthesized complementary
sister strand by a DNA-dependent DNA polymerase.
[0113] In another aspect, the term "encode" refers to any process
whereby the information in one molecule is used to direct the
production of a second molecule that has a different chemical
nature from the first molecule. For example, a DNA molecule can
encode an RNA molecule (for instance, by the process of
transcription incorporating a DNA-dependent RNA polymerase enzyme).
Also, an RNA molecule can encode a peptide, as in the process of
translation. When used to describe the process of translation, the
term "encode" also extends to the triplet codon that encodes an
amino acid. In some aspects, an RNA molecule can encode a DNA
molecule, for instance, by the process of reverse transcription
incorporating an RNA-dependent DNA polymerase. In another aspect, a
DNA molecule can encode a peptide, where it is understood that
"encode" as used in that case incorporates both the processes of
transcription and translation.
[0114] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acid consists of nitrogenous bases that are
either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or
purines (adenine (A) and guanine (G)). These nitrogenous bases form
hydrogen bonds between a pyrimidine and a purine, and the bonding
of the pyrimidine to the purine is referred to as "base pairing."
More specifically, A will hydrogen bond to T or U, and G will bond
to C. "Complementary" refers to the base pairing that occurs
between two distinct nucleic acid sequences or two distinct regions
of the same nucleic acid sequence. For example, an oligonucleotide
can be complementary to a prion protein-encoding RNA, or a prion
protein-encoding DNA.
[0115] "Specifically hybridizable" and "specifically complementary"
are terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the oligonucleotide
(or its analog) and the DNA or RNA target. The oligonucleotide or
oligonucleotide analog need not be 100% complementary to its target
sequence to be specifically hybridizable. An oligonucleotide or
analog is specifically hybridizable when binding of the
oligonucleotide or analog to the target DNA or RNA molecule
interferes with the normal function of the target DNA or RNA, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide or analog to non-target
sequences under conditions where specific binding is desired, for
example under physiological conditions in the case of in vivo
assays or systems. Such binding is referred to as specific
hybridization.
[0116] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na+ and/or Mg++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are
discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.
[0117] In a particular example, stringent conditions are
hybridization at 65.degree. C. in 6.times.SSC, 5.times.Denhardt's
solution, 0.5% SDS and 100 .mu.g sheared salmon testes DNA,
followed by 15 30-minute sequential washes at 65.degree. C. in
2.times.SSC, 0.5% SDS, followed by 1.times.SSC, 0.5% SDS and
finally 0.2.times.SSC, 0.5% SDS.
[0118] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, peptide, or cell) has been purified away
from other biological components in a mixed sample (such as a cell
extract). For example, an "isolated" peptide or nucleic acid
molecule is a peptide or nucleic acid molecule that has been
separated from the other components of a cell in which the peptide
or nucleic acid molecule was present (such as an expression host
cell for a recombinant peptide or nucleic acid molecule).
[0119] Nucleic acid molecule: A polymeric form of nucleotides,
which can include both sense and anti sense strands of RNA, cDNA,
genomic DNA, and synthetic forms and mixed polymers of the above. A
nucleotide refers to a ribonucleotide, deoxynucleotide or a
modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." A nucleic acid molecule is usually at least 10
bases in length, unless otherwise specified. The term includes
single and double stranded forms of DNA. A nucleic acid molecule
can include either or both naturally occurring and modified
nucleotides linked together by naturally occurring and/or non
naturally occurring nucleotide linkages.
[0120] Prion: a type of infectious agent composed mainly of
protein. Prions cause a number of diseases in a variety of animals,
including bovine spongiform encephalopathy (BSE, also known as mad
cow disease) in cattle and Creutzfeldt-Jakob disease in humans. All
known prion diseases affect the structure of the brain or other
neural tissue, and all are untreatable and fatal.
[0121] Prions are believed to infect and propagate by refolding
abnormally into a structure that is able to convert normal
molecules of the protein into the abnormally structured (for
instance, PrP-res or Prp.sup.Sc) form. Most, if not all, known
prions can polymerize into amyloid fibrils rich in tightly packed
beta sheets. This altered structure renders them unusually
resistant to denaturation by chemical and physical agents, making
disposal and containment of these particles difficult.
[0122] In prion diseases, the pathological, protease-resistant form
of prion protein, termed PrP.sup.Sc or PrP-res, appears to
propagate itself in infected hosts by inducing the conversion of
its normal host-encoded protease-sensitive precursor, PrP-sen, into
PrP.sup.Sc. PrP-sen is a monomeric glycophosphatidylinositol-linked
glycoprotein that is low in .beta.-sheet content, and highly
protease-sensitive. Conversely, PrP.sup.Sc aggregates are high in
.beta.-sheet content and partially protease-resistant. Mechanistic
details of the conversion are not well understood, but involve
direct interaction between PrP.sup.Sc and PrP-sen, resulting in
conformational changes in PrP-sen as the latter is recruited into
the growing PrP.sup.Sc multimer (reviewed in Caughey & Baron
(2006) Nature 443, 803-810). Accordingly, the conversion mechanism
has been tentatively described as autocatalytic seeded (or
nucleated) polymerization.
[0123] PMCA or Protein Misfolding Cyclic Amplification: A method
for amplifying PrP-res in a sample by mixing Prp-sen with the
sample, incubating the reaction mix to permit PrP-res to initiate
the conversion of PrP-sen to aggregates of PrP-res, fragmenting any
aggregates formed during the incubation step (typically by
sonication), and repeating one or more cycles of the incubation and
fragmentation steps.
[0124] QUIC or Quaking Induced Conversion: A particular type of
rPrP-sen amplification assay, in which shaking of the reaction
vessels is performed instead of sonication to disrupted aggregated
PrP-sen and PrP-res.
[0125] Sequence identity: The similarity between two nucleic acid
sequences or between two amino acid sequences is expressed in terms
of the level of sequence identity shared between the sequences.
Sequence identity is typically expressed in terms of percentage
identity; the higher the percentage, the more similar the two
sequences. Methods for aligning sequences for comparison are
described in detail below, in section IV E of the Detailed
Description.
[0126] Single Round: Performing a method wherein serial
amplification is not performed. For example, PrP-res can be
amplified in a sample, by mixing the sample with purified rPrP-sen
to make a reaction mix; performing an amplification reaction that
includes (i) incubating the reaction mix to permit coaggregation of
the rPrP-sen with the PrP-res that may be present in the reaction
mix, and maintaining incubation conditions that promote
coaggregation of the rPrP-sen with the PrP-res and results in a
conversion of the rPrP-sen to rPrP-res.sup.(Sc) while inhibiting
development of rPrP-res.sup.(spon); (ii) agitating aggregates
formed during step (i); (iii) optionally repeating steps (i) and
(ii) one or more times. rPrP-res.sup.(Sc) is detected in the
reaction mix, wherein detection of rPrP-res.sup.(Sc) in the
reaction mix indicates that PrP-res was present in the sample.
However, a portion of the reaction mix is not removed and incubated
with additional rPrP-sen.
[0127] Suitable methods and materials for the practice or testing
of the disclosure are described below. However, the provided
materials, methods, and examples are illustrative only and are not
intended to be limiting. Accordingly, except as otherwise noted,
the methods and techniques of the present disclosure can be
performed according to methods and materials similar or equivalent
to those described and/or according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification (see, for instance, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel
et al., Current Protocols in Molecular Biology, Greene Publishing
Associates, 1992 (and Supplements to 2000); Ausubel et al., Short
Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, 4th ed., Wiley & Sons,
1999).
IV. Detailed Description of Particular Embodiments
[0128] A. Overview of Prions and Prion Disease
[0129] The transmissible spongiform encephalopathies (TSEs, or
prion diseases) are infectious neurodegenerative diseases of
mammals that include (but are not limited to) scrapie in sheep,
bovine spongiform encephalopathy (BSE; also known as mad cow
disease) in cattle, transmissible mink encephalopathy (TME) in
mink, chronic wasting disease (CWD) in elk, moose, and deer, feline
spongiform encephalopathy in cats, exotic ungulate encephalopathy
(EUE) in nyala, oryx and greater kudu, and Creutzfeldt-Jakob
disease (CJD) and its varieties (iatrogenic Creutzfeldt-Jakob
disease (iCJD), variant Creutzfeldt-Jakob disease (vCJD), familial
Creutzfeldt-Jakob disease (fCJD), and sporadic Creutzfeldt-Jakob
disease (sCJD)), Gerstmann-Straussler-Scheinker syndrome (GSS),
fatal familial insomnia (fFI), sporadic fatal insomnia (sFI), kuru,
and Alpers syndrome in humans. TSEs have incubation periods of
months to years, but after the appearance of clinical signs often
are rapidly progressive, untreatable, and invariably fatal.
Attempts at TSE risk reduction have led to profound changes in the
production and trade of agricultural goods, medicines, cosmetics,
and biotechnology products.
[0130] In TSEs the pathological, protease-resistant form of prion
protein, termed PrP.sup.Sc or PrP-res, appears to propagate itself
in infected hosts by inducing the conversion of its normal
host-encoded precursor, PrP-sen, into PrP.sup.Sc. PrP-sen is a
monomeric glycophosphatidylinositol-linked glycoprotein that is low
in .beta.-sheet content, and highly protease-sensitive. Conversely,
PrP.sup.Sc aggregates are high in .beta.-sheet content and
partially protease-resistant. Mechanistic details of the conversion
are not well understood, but involve direct interaction between
PrP.sup.Sc and PrP-sen, resulting in conformational changes in
PrP-sen as the latter is recruited into the growing PrP.sup.Sc
multimer (reviewed in Caughey & Baron (2006) Nature 443,
803-810). Accordingly, the conversion mechanism has been
tentatively described as autocatalytic seeded (or nucleated)
polymerization.
[0131] To better understand the mechanism of prion propagation,
many attempts to recapitulate PrP.sup.Sc formation in cell-free
systems have been made. Initial experiments showed that PrP.sup.Sc
can induce the conversion of PrP-sen to PrP.sup.Sc with strain- and
species-specificities, albeit with substoichiometric yields. More
recently, it was shown that PrP.sup.Sc formation and TSE
infectivity can be amplified indefinitely in crude brain
homogenates, a medium containing numerous potential cofactors for
conversion (Castilla et al., (2005) Cell 121, 195-206). Dissection
of this "protein misfolding cyclic amplification" (PMCA) reaction
showed that PrP.sup.Sc and prion infectivity also could be
amplified using PrP-sen purified from brain tissue as long as
polyanions such as RNA were added (Deleault et al., (2007) Proc
Natl Acad Sci USA. 104(23):9741-6). Recombinant PrP-sen (rPrP-sen)
from E. coli lacks glycosylation and the GPI anchor and prior to
this disclosure has not been used successfully as an amplification
substrate in PrP.sup.Sc-seeded PMCA reactions. In fact, it was
previously reported that rPrP-sen does not work in the PMCA system
(Nishina et al., (2006) Biochemistry 45(47):14129-39). However,
rPrP-sen can be converted to protease-resistant forms with limited
yields when mixed with PrP.sup.Sc. rPrP-sen also can be induced to
polymerize into amyloid fibrils spontaneously or when seeded by
preformed rPrP fibrils. Although most rPrP amyloid preparations are
not infectious, synthetic amyloid fibrils of mutant recombinant
prion protein can cause or accelerate TSE disease in transgenic
mice that vastly overexpress the same mutant prion protein
construct (Legname et al. (2004) Science 305, 673-676). However,
these "synthetic prions" were non-infectious for wild type mice,
making them at least 10.sup.8-fold less infectious than bona fide
PrP.sup.8c. Thus, the basic structure and propagation mechanism of
robust TSE infectivity (or prions) remains to be fully
ascertained.
[0132] A key challenge in coping with TSEs is the rapid detection
of low levels of TSE infectivity (prions) by rapid methods. The
most commonly used marker for TSE infections is PrP.sup.8c, and the
PMCA reaction allows extremely sensitive detection of PrP.sup.Sc at
levels below single infectious units in infected tissue. However,
as previously noted, current limitations of PMCA include the time
required to achieve optimal sensitivity (.about.3 weeks) and the
use of brain PrP-sen as the amplification substrate.
[0133] B. Transmissible Spongiform Encephalopathies (TSEs)
[0134] The most common TSE in animals is scrapie, but the most
famous and dangerous TSE is BSE, which affects cattle and is known
by its lay term "mad cow disease." In humans, the most common TSE
is CJD, which occurs worldwide with an incidence of 0.5 to 1.5 new
cases per one million people each year. Three different forms of
CJD have been traditionally recognized: sporadic (sCJD; 85% of
cases), familial (fCJD; 10%), and iatrogenic (iCJD; 5%). However,
in 1996, a new variant form of CJD (vCJD) emerged in the UK that
was associated with consumption of meat infected with BSE. In
contrast with typical sCJD, vCJD affects young patients with an
average age of 27 years, and causes a relatively long illness (14
months compared with 4.5 months for sCJD). Because of insufficient
information available about the incubation time and the levels of
exposure to contaminated cattle food products, it is difficult to
predict the future incidence of vCJD. In animals, there is no
evidence for inherited forms of the disease, and most cases appear
to be acquired by horizontal or vertical transmission.
[0135] The clinical diagnosis of sCJD is based on a combination of
rapidly progressive multifocal dementia with pyramidal and
extrapyramidal signs, myoclonus, and visual or cerebellar signs,
associated with a characteristic periodic electroencephalogram
(EEG). A key diagnostic feature of sCJD that distinguishes it from
Alzheimer's disease and other dementias is the rapid progression of
clinical symptoms and the short duration of the disease, which is
often less than 2 years. The clinical manifestation of fCJD is very
similar, except that the disease onset is slightly earlier than in
sCJD. Family history of inherited CJD or genetic screening for
mutations in the prion protein gene are used to establish fCJD
diagnosis, although lack of family history does not excludes an
inherited origin.
[0136] Variant CJD appears initially as a progressive
neuropsychiatric disorder characterized by symptoms of anxiety,
depression, apathy, withdrawal and delusions, combined with
persistent painful sensory symptoms and followed by ataxia,
myoclonus, and dementia. Variant CJD is differentiated from sCJD by
the duration of illness (usually longer than 6 months) and EEG
analysis (vCJD does not show the atypical pattern observed in
sCJD). A high bilateral pulvinar signal noted during MRI is often
used to help diagnose vCJD. In addition, a tonsil biopsy can be
used to help diagnose vCJD, based on a number of cases of vCJD have
been shown to test positive for PrPSc staining in lymphoid tissue
(such as tonsil and appendix). However, because of the invasive
nature of this test, it is performed only in patients who fulfill
the clinical criteria of vCJD where the MRI of the brain does not
show the characteristic pulvinar sign.
[0137] GSS is a dominantly inherited illness that is characterized
by dementia, Parkinsonian symptoms, and a relatively long duration
(typically, 5-8 years). Clinically, GSS is similar to Alzheimer's
disease, except that is often accompanied by ataxia and seizures.
Diagnosis is established by clinical examination and genetic
screening for prion protein mutations. FFI is also dominantly
inherited and associated with prion protein mutations. However, the
major clinical finding associated with FPI is insomnia, followed at
late stages by myoclonus, hallucinations, ataxia, and dementia.
[0138] C. Protein Misfolding Cyclic Amplification (PMCA), and
rPrP-Res Amplification (rPrP-PMCA and QUIC)
[0139] The prion detection method termed protein misfolding cyclic
amplification (PMCA) is based on the ability of prions to replicate
in vitro in tissue homogenates containing PrP-sen (see, for
instance, WO0204954). PMCA involves amplification of a PrP-res
through incubation with a suitable prion protein substrate derived
from brain tissue, serial amplification of the PrP-res, for
instance by alternating incubation and sonication steps, and
detection of the resulting PrP-res.sup.(Sc). In some instances,
incubation and sonication are alternated over a period of
approximately three weeks, and intermittently a portion of the
reaction mix is removed and incubated with additional PrP-sen in
order to serially amplify the PrP-res in the sample. Following the
repeated incubation/sonication/dilution steps, the resulting
PrP-res.sup.(Sc) is detected in the reaction mix. Although PMCA is
a very sensitive assay for detecting PrP-res, it has a number of
limitations, notably the time required to achieve optimal
sensitivity (.about.3 weeks) and the requirement for brain-derived
PrP-sen as the amplification substrate.
[0140] The development of more sensitive, rapid, and practical
means for detection of PrP.sup.Sc and TSE infectivity is critical
in addressing the challenges posed by prion diseases. Such a test
could be used to identify sources of TSE infection in agriculture
and the environment to reduce risks to humans and animals.
Moreover, the ability to diagnose infections in humans long before
the appearance of clinical signs would greatly improve the chances
of treating these otherwise fatal diseases. Indeed, drug treatments
in animals tend to be much more effective when treatments are
initiated within the first two thirds of the incubation period
Caughey et al. (2006) Accts. Chem. Res. 39, 646-653; Trevitt &
Collinge (2006) Brain 129, 2241-2265).
[0141] Disclosed herein is an improved prion assay, termed rPrP-res
amplification assay (including rPrP-PMCA and QUIC), that differs
from the PMCA PrP.sup.Sc amplification method (Saa et al., (2006)
J. Biol. Chem. 281, 35245-35252; Saa et al., (2006) Science 313,
92-94). rPrP-PMCA greatly improves the practicality of the basic
PMCA approach in several significant ways. First, instead of prion
protein substrate derived from brain tissue, rPrP-PMCA and QUIC
(when agitation is performed by shaking) makes use of
bacterially-expressed rPrP-sen as a substrate, which can be
obtained rapidly in high purity and in large amounts, whereas
purification of PrP-sen from brain tissue is difficult and gives
much lower yields (Deleault et al. (2005) J. Biol. Chem. 280,
26873-26879; Pan et al. (1993) Proc. Natl. Acad. Sci. USA 90,
10962-10966; Hornemann et al., (2004) EMBO Rep. 5, 1159-1164).
Furthermore, unlike PrP-sen in brain homogenates or purified from
brain, rPrP-sen can be easily mutated or strategically labeled with
probes to simplify and accelerate the detection of relevant
rPrP-PMCA products.
[0142] There are two types of rPrP-res amplification methods that
utilize rPrP-sen, one that uses sonication (rPrP-PMCA) and one that
utilizes shaking (QUIC). These methods facilitate fundamental
studies of the structure and conversion mechanism of PrP.sup.Sc.
Site-directed mutations can allow precise labeling of rPrP-sen with
a variety of probes that can report on conformational changes, and
both inter-molecular and intra-molecular distances within rPrP-res
aggregates.
[0143] The rPrP-PMCA and QUIC methods generally involve mixing a
sample (for example a tissue sample or CSF sample that is suspected
of containing PrP-res) with purified rPrP-sen to make a reaction
mix, and performing a primary reaction to form and amplify specific
forms of rPrP-res in the mixture. This primary reaction includes
incubating the reaction mix to permit the PrP-res to initiate the
conversion of rPrP-sen to specific aggregates or polymers of
rPrP-res; fragmenting any aggregates or polymers formed during the
incubation step; and repeating the incubation and fragmentation
steps one or more times, for instance from about 10 to about 50
times. In some embodiments of the method, serial amplification is
carried out by removing a portion of the reaction mix and
incubating it with additional rPrP-sen. Following amplification,
the prion-initiated rPrP-res.sup.(Sc) in the reaction mix is
detected, for example using an antibody. In some examples, the
reaction mix is digested with proteinase K (which digests the
remaining rPrP-sen in the reaction mix) prior to detection of the
rPrP-res.sup.(Sc). Two types of mis-folded prion protein can be
generated in rPrP-PMCA (or QUIC) reactions, one occurring
spontaneously (rPrP-res.sup.(spon)) and the other initiated by the
presence of prions (rPrP-res.sup.(Sc)) in the test sample. Thus, it
is often necessary to discriminate between the former and the
latter to interpret the rPrP-PMCA assay. For instance, this can be
done on the basis of differing protein fragment sizes generated
upon exposure to proteinase K. An unexpectedly superior decrease in
the amount of rPrP-res.sup.(spon)) formed is achieved with the QUIC
assay.
[0144] The use of recombinant prion protein as a substrate for the
QUIC reaction instead of PrP-sen contained in, or isolated from,
brain homogenates (which is the source of substrate in conventional
PMCA) confers several advantages. For instance, successful
expression and folding of rPrP enables the generation of large
amounts of highly purified and concentrated substrate, which is not
possible when the only available source of substrate is brain
tissue. Additionally and surprisingly, the use of concentrated
recombinant prion protein promotes far faster amplification
reactions than does PrP-sen in brain homogenate. It is this
surprising functionality of the rPrP that reduces the time required
for the reaction from up to three weeks to about 1-2 days, or even
less than a day.
[0145] All of the methods disclosed herein, such as QUIC, will work
under a variety of conditions. In several embodiments, optimal
conditions that support specific PrP.sup.Sc-seeded QUIC include the
use of a detergent, such as both an ionic and a non-ionic
detergent. The conditions can include the combination of about
0.05-0.1% of an ionic detergent such as SDS and about 0.05-0.1% of
a nonionic detergent such as TX-100 in the reaction mix. Other
preferred conditions include the use of shaking instead of
sonication (the so-called QUIC reaction), and the use of cycles of
shaking/rest that are about 1:1 in duration. Reactions have also
been found to be particularly efficient at 37-60.degree. C., for
example 45-55.degree. C. These conditions are particularly
effective at promoting the formation of rPrP-res.sup.(Sc) (notably
the 17 kDa PK-resistant species), while reducing
rPrP-res.sup.(spon) formation within the first 24 hours of unseeded
reactions. However, longer amplification reactions of more than 24
hours, such as at least 45 hours or even 65 or 96 hours, can also
provide excellent results.
[0146] The sensitivity of the assay has been found to be degraded
(and potential false positive results are obtained) by the
production of rPrP-res.sup.(spon) in the use of rPrP-sen seeded
reactions. To help avoid this problem, conditions are selected to
inhibit the formation of the rPrP-res.sup.(spon) byproduct. In some
examples, assays (such as QUIC) are performed to test assay
conditions to determine if the assay conditions increase or
decrease rPrP-res.sup.(spon) byproduct formation, and assay
conditions are selected that minimize the byproduct formation. The
recognition of this previously unappreciated obstacle to the use of
amplification assays has also helped provide a much faster and more
sensitive assay to address this substantial public health
concern.
[0147] The sensitivity achieved with rPrP-PMCA (and QUIC) is of
considerable utility because it is very sensitive. For example, the
assay allows consistent detection of HaPrP.sup.Sc levels (50 ag)
that are >100-fold lower than those typically associated with a
lethal intracerebral dose of 263K strain scrapie infectivity in
Syrian golden hamsters. Although this detection limit is not quite
as low as that reported for the conventional PMCA (1.2 ag
PrP.sup.Sc; Saa et al., (2006) J. Biol. Chem. 281, 35245-35252), it
can be achieved in two rPrP-PMCA rounds of amplification over a
total of about two days, whereas conventional PMCA requires seven
rounds over a total of about 21 days (Saa et al., (2006) J. Biol.
Chem. 281, 35245-35252). A single 50-hour round of conventional
PMCA takes about the same time as two rounds of rPrP-PMCA, but has
a 32,000-fold higher detection limit (1.6 pg; Saa et al., (2006) J.
Biol. Chem. 281, 35245-35252). Without being bound by theory, it is
believed that the more rapid rPrP-PMCA reaction is facilitated
in-part by the higher concentration of rPrP-sen relative to that of
PrP-sen in brain homogenates.
[0148] It has also been found that the rPrP-PMCA/QUIC assay can
perform cross-species amplification of target PrP-res. In fact,
rHaPrP-PMCA/QUIC provides a particularly suitable form of rPrP-res
that promotes formation of PrP aggregates when incubated with a
sample that contains PrP-res. rHaPrP appears to have a structure
that promotes the formation of these aggregates with minimal
formation of rPrP-res.sup.(spon) byproduct. Hence rHaPrP can be
used to amplify target PrP in a sample taken from a species other
than a hamster, such as a sample taken from a human, sheep, cow or
cervid.
[0149] Another advantage of the rPrP-PMCA and QUIC assays is the
ability to discriminate between scrapie-infected and uninfected
hamsters using 2-.mu.l CSF samples (see FIG. 4). Because CSF is
more accessible in live individuals than is brain tissue, it is an
attractive biopsy specimen for rPrP-PMCA- and QUIC-based diagnostic
tests.
[0150] D. Recombinant Prion Protein
[0151] As described herein, the PrP-sen in used in rPrP-res PMCA
reaction is recombinant prion protein, for example prion protein
from cells engineered to over express the protein. Any prion
protein sequence can be used to generate the rPrP-sen, for
instance: Xenopus laevis (Genbank Accession No: NP001082180), Bos
Taurus (Genbank Accession No: CAA39368), Danio verio (Genbank
Accession No: NP991149), Tragelaphus strepsiceros (Genbank
Accession No: CAA52781), Ovis aries (Genbank Accession No:
CAA04236), Trachemys scripta (Genbank Accession No: CAB81568),
Gallus gallus (Genbank Accession No: AAC28970), Rattus norvegicus
NP036763), Mus musculus (Genbank Accession No: NP035300),
Monodelphis domestica (Genbank Accession No: NP001035117), Homo
sapiens (Genbank Accession No: BAA00011), Giraffa camelopardalis
(Genbank Accession No: AAD13290), Oryctolagus cuniculus (Genbank
Accession No: NP001075490), Macaca mulatta (Genbank Accession No:
NP001040617), Bubalus bubalus (Genbank Accession No: AAV30514),
Tragelaphus imberbis (Genbank Accession No: AAV30511), Boselaphus
tragocamelus (Genbank Accession No: AAV30507), Bos garus (Genbank
Accession No: AAV 30505), Bison bison (Genbank Accession No:
AAV30503), Bos javanicus (Genbank Accession No: AAV30498), Syncerus
caffer caffer (Genbank Accession No: AAV30492), Syncerus caffer
nanus (Genbank Accession No: AAV30491), and Bos indicus (Genbank
Accession No: AAV30489). In some embodiments, only a partial prion
protein sequence is expressed as rPrP-sen. For instance, in certain
examples rPrP-sen includes amino acids 23-231 (SEQ ID NOS: 1, 2) of
the hamster (SEQ ID NO: 8) or mouse (SEQ ID NO: 9) prion protein
sequences, or the corresponding amino acids of other prion protein
sequences, for instance amino acids 23-231 (SEQ ID NO: 3) of human
(129M) prion protein (SEQ ID NO: 10), amino acids 23-231 (SEQ ID
NO: 4) of human (129V) prion protein (SEQ ID NO: 11), amino acids
25-241 (SEQ ID NO: 5) of bovine (6-octarepeat) prion protein, amino
acids 25-233 (SEQ ID NO: 6) of ovine (136A 154R 171Q) prion
protein, or amino acids 25-234 (SEQ ID NO: 7) of deer (96G 132M
138S) prion protein. In general, the partial prion protein sequence
expressed as rPrP-sen corresponds to the polypeptide sequences of
the natural mature full-length PrP c molecule, meaning that the
rPrP-sen polypeptide lacks both the amino-terminal signal sequence
and carboxy-terminal glycophosphatidylinositol-anchor attachment
sequence. In another example, amino acids 30-231, 40-231, 50-231,
60-231, 70-231, 80-231, or 90-231 of any one of human, human 129V,
bovine, ovine, or deer are utilized in the assays described herein.
One of skill in the art can readily produces these polypeptides
using the sequence information provided in SEQ ID NOs: 1-11, or
using information available in GENBANK.RTM. (as available on Jul.
20, 2007).
[0152] The rPrP-sen can be a chimeric rPrP-sen, wherein a portion
of the protein is from one species, and a portion of the protein is
from another species, can also be utilized. In one example about 10
to about 90%, such as about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70% about 80% or about 90% of the
rPrP-sen is from one species, and, correspondingly, about 90%,
about 80%, about 70%, about 60%, about 50%, about 40%, about 30%,
about 20% or about 10% is from another species. Chimeric proteins
can include, for example, hamster rPrP-sen and rPrP-sen from
another species, such as human PrP-sen.
[0153] In some embodiments, host cells are transformed with a
nucleic acid vector that expresses the rPrP-sen, for example human,
cow, sheep or hamster rPrP-sen. These cells can be mammalian cells,
bacterial cells, yeast cells, insect cells, whole organisms, such
as transgenic mice, or other cells that can serve as source of the
PrP-sen. In particular examples the cell is a bacterial cell, such
as an E. coli cell. Raw cell lysates or purified rPrP-sen from
rPrP-sen expressing cells can be used as the source of the
non-pathogenic protein.
[0154] In some embodiments the recombinant protein is fused with an
additional amino acid sequence. For example, over expressed protein
can be tagged for purification or to facilitate detection of the
protein in a sample. Some possible fusion proteins that can be
generated include histidine tags, Glutathione 5-transferase (GST),
Maltose binding protein (MBP), green fluorescent protein (GFP), and
Flag and myc-tagged rPrP. These additional sequences can be used to
aid in purification and/or detection of the recombinant protein,
and in some cases are subsequently removed by protease cleavage.
For example, coding sequence for a specific protease cleavage site
can be inserted between the PrP-sen coding sequence and the
purification tag coding sequence. One example for such a sequence
is the cleavage site for thrombin. Thus, fusion proteins can be
cleaved with the protease to free the PrP-sen from the purification
tag.
[0155] Any of the wide variety of vectors known to those of skill
in the art can be used to over-express rPrP-sen. For example,
plasmids or viral vectors can be used. These vectors can be
introduced into cells by a variety of methods including, but not
limited to, transfection (for instance, by liposome, calcium
phosphate, electroporation, particle bombardment, and the like),
transformation, and viral transduction.
[0156] Recombinant PrP-sen also can include proteins that have
amino sequences containing substitutions, insertions, deletions,
and stop codons as compared to wild type sequences. In certain
embodiment, a protease cleavage sequence is added to allow
inactivation of protein after it is converted into prion form. For
example, cleavage sequences recognized by Thrombin, Tobacco Etch
Virus (Life Technologies, Gaithersburg, Md.) or Factor Xa (New
England Biolabs, Beverley, Mass.) proteases can be inserted into
the sequence. In some embodiments, inactivation of protein after it
is converted into prion form is unnecessary because the
rPrP-res.sup.(Sc) resulting from the reaction has little or no
infectivity.
[0157] Changes also can be made in the pPrP-sen protein coding
sequence, for example in the coding sequence for mouse, human,
bovine, sheep, goat, deer and/or elk prion protein (GENBANK.RTM.
accession numbers NM_011170, NM_183079, AY335912, AY723289,
AY723292, AF156185 and AY748455, respectively, all of which are
incorporated herein by reference, Jul. 20, 2007). For example,
mutations can be made to match a variety of mutations and
polymorphisms known for various mammalian prion protein genes (see,
for instance, Table 1). Furthermore, chimeric PrP molecules
comprising sequences from two or more different natural PrP
sequences (for instance from different host species or strains) can
be expressed from vectors with recombinant PrP gene sequences, and
such chimeras can be used for rPrP-PMCA and QUIC detection of prion
from various species. Cells expressing these altered prion protein
genes can be used as a source of the rPrP-sen, and these cells can
include cells that endogenously express the mutant rPrP gene, or
cells that have been made to express a mutant rPrP protein by the
introduction of an expression vector. Use of a mutated rPrP-sen can
be advantageous, because some of these proteins are more easily or
specifically converted to protease-resistant forms, and thus can
further enhance the sensitivity of the method.
[0158] In certain embodiments, cysteine residues are placed at
positions 94 and 95 of the hamster prion protein sequence in order
to be able to selectively label the rPrP at those sites using
sulfhydryl-reactive labels, such as pyrene and fluorescein linked
to maleimide-based functional groups. In certain embodiments, these
tags do not interfere with conversion but allow much more rapid,
fluorescence-based detection of the rPrP-PMCA reaction product. In
one example, pyrenes in adjacent molecules of rPrP-res are held in
close enough proximity to allow eximer formation, which shifts the
fluorescence emission spectrum in a distinct and detectable manner
Free pyrenes released from, or on, unconverted rPrP-sen molecules
are unlikely to form eximer pairs. Thus, the rPrP-PMCA reaction can
be run in a multiwell plate, digested with proteinase K, and then
eximer fluorescence can be measured to rapidly test for the
presence of rPrP-res.sup.(Sc). Sites 94 and 95 were chosen for the
labels because the PK-resistance in this region of PrP-res
distinguishes rPrP-res.sup.(Sc) from rPrP-res.sup.(spon), giving
rise to the 17 kDa rPrP-res band. Other positions in the
PK-resistant region(s) that distinguish the 17-kDa
rHaPrP-res.sup.(Sc) fragment from all rHaPrP-res.sup.(spon)
fragments also can work for this purpose.
TABLE-US-00007 TABLE 1 Pathogenic human Human Ovine Bovine
mutations polymorphisms polymorphisms polymorphisms 2 octarepeat
insert Codon 129 Codon 171 5 or 6 4-9 octarepeat insert Met/Val
Arg/Glu octarepeats Codon 102 Pro-Leu Codon 219 Codon 136 Codon 105
Pro-Leu Glu/Lys Ala/Val Codon 117 Ala-Val Codon 145 Stop Codon 178
Asp-A Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon
210 Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala
[0159] E. Variant Prion Protein Sequences
[0160] As any molecular biology textbook teaches, a peptide of
interest is encoded by its corresponding nucleic acid sequence (for
instance, an mRNA or genomic DNA). Accordingly, nucleic acid
sequences encoding prion proteins are contemplated herein, at
least, to make and use the prion proteins of the disclosed
compositions and methods.
[0161] In one example, in vitro nucleic acid amplification (such as
polymerase chain reaction (PCR)) can be utilized as a method for
producing nucleic acid sequences encoding prion proteins. PCR is a
standard technique that is described, for instance, in PCR
Protocols: A Guide to Methods and Applications (Innis et al., San
Diego, Calif.: Academic Press, 1990), or PCR Protocols, Second
Edition (Methods in Molecular Biology, Vol. 22, ed. by Bartlett and
Stirling, Humana Press, 2003).
[0162] A representative technique for producing a nucleic acid
sequence encoding a prion protein by PCR involves preparing a
sample containing a target nucleic acid molecule that includes the
prion protein-encoding sequence. For example, DNA or RNA (such as
mRNA or total RNA) can serve as a suitable target nucleic acid
molecule for PCR reactions. Optionally, the target nucleic acid
molecule can be extracted from cells by any one of a variety of
methods well known to those of ordinary skill in the art (for
instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel et
al., Current Protocols in Molecular Biology, Greene Publ. Assoc.
and Wiley-Intersciences, 1992). Prion proteins are expressed in a
variety of mammalian cells. In examples where RNA is the initial
target, the RNA is reverse transcribed (using one of a myriad of
reverse transcriptases commonly known in the art) to produce a
double-stranded template molecule for subsequent amplification.
This particular method is known as reverse transcriptase (RT)-PCR.
Representative methods and conditions for RT-PCR are described, for
example, in Kawasaki et al. (In PCR Protocols, A Guide to Methods
and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc.,
San Diego, Calif., 1990).
[0163] The selection of amplification primers will be made
according to the portion(s) of the target nucleic acid molecule
that is to be amplified. In various embodiments, primers
(typically, at least 10 consecutive nucleotides of prion-encoding
nucleic acid sequence) can be chosen to amplify all or part of a
prion-encoding sequence. Variations in amplification conditions may
be required to accommodate primers and amplicons of differing
lengths and composition; such considerations are well known in the
art and are discussed for instance in Innis et al. (PCR Protocols,
A Guide to Methods and Applications, San Diego, Calif.: Academic
Press, 1990). From a provided prion protein-encoding nucleic acid
sequence, one skilled in the art can easily design many different
primers that can successfully amplify all or part of a prion
protein-encoding sequence.
[0164] As described herein, a number of prion protein-encoding
nucleic acid sequences are known. Though particular nucleic acid
sequences are disclosed, one of skill in the art will appreciate
that also provided are many related sequences with the functions
described herein, for instance, nucleic acid molecules encoding
conservative variants of a prion protein. One indication that two
nucleic acid molecules are closely related (for instance, are
variants of one another) is sequence identity, a measure of
similarity between two nucleic acid sequences or between two amino
acid sequences expressed in terms of the level of sequence identity
shared between the sequences. Sequence identity is typically
expressed in terms of percentage identity; the higher the
percentage, the more similar the two sequences.
[0165] Methods for aligning sequences for comparison are well known
in the art. Various programs and alignment algorithms are described
in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and
Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl.
Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244,
1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al.,
Nucleic Acids Research 16:10881-10890, 1988; Huang, et al.,
Computer Applications in the Biosciences 8:155-165, 1992; Pearson
et al., Methods in Molecular Biology 24:307-331, 1994; Tatiana et
al., (1999), FEMS Microbiol. Lett., 174:247-250, 1999. Altschul et
al. present a detailed consideration of sequence-alignment methods
and homology calculations (J. Mol. Biol. 215:403-410, 1990).
[0166] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM., Altschul et al., J.
Mol. Biol. 215:403-410, 1990) is available from several sources,
including the National Center for Biotechnology Information (NCBI,
Bethesda, Md.) and on the Internet, for use in connection with the
sequence-analysis programs blastp, blastn, blastx, tblastn and
tblastx. A description of how to determine sequence identity using
this program is available on the internet under the help section
for BLAST.TM..
[0167] For comparisons of amino acid sequences of greater than
about 30 amino acids, the "Blast 2 sequences" function of the
BLAST.TM. (Blastp) program is employed using the default BLOSUM62
matrix set to default parameters (cost to open a gap [default=5];
cost to extend a gap [default=2]; penalty for a mismatch
[default=-3]; reward for a match [default=1]; expectation value (E)
[default=10.0]; word size [default=3]; number of one-line
descriptions (V) [default=100]; number of alignments to show (B)
[default=100]). When aligning short peptides (fewer than around 30
amino acids), the alignment should be performed using the Blast 2
sequences function, employing the PAM30 matrix set to default
parameters (open gap 9, extension gap 1 penalties). Proteins with
even greater similarity to the reference sequences will show
increasing percentage identities when assessed by this method, such
as at least 50%, at least 60%, at least 70%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98%, or at least 99%
sequence identity to the sequence of interest.
[0168] For comparisons of nucleic acid sequences, the "Blast 2
sequences" function of the BLAST.TM. (Blastn) program is employed
using the default BLOSUM62 matrix set to default parameters (cost
to open a gap [default=11]; cost to extend a gap [default=1];
expectation value (E) [default=10.0]; word size [default=11];
number of one-line descriptions (V) [default=100]; number of
alignments to show (B) [default=100]). Nucleic acid sequences with
even greater similarity to the reference sequences will show
increasing percentage identities when assessed by this method, such
as at least 60%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, r at least 98%, or at least 99%
sequence identity to the prion sequence of interest.
[0169] Another indication of sequence identity is nucleic acid
hybridization. In certain embodiments, prion protein-encoding
nucleic acid variants hybridize to a disclosed (or otherwise known)
prion protein-encoding nucleic acid sequence, for example, under
low stringency, high stringency, or very high stringency
conditions. Hybridization conditions resulting in particular
degrees of stringency will vary depending upon the nature of the
hybridization method of choice and the composition and length of
the hybridizing nucleic acid sequences. Generally, the temperature
of hybridization and the ionic strength (especially the Nat
concentration) of the hybridization buffer will determine the
stringency of hybridization, although wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are
discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.
[0170] The following are representative hybridization conditions
and are not meant to be limiting.
Very High Stringency (Detects Sequences that Share at Least 90%
Sequence Identity) Hybridization: 5.times.SSC at 65.degree. C. for
16 hours Wash twice: 2.times.SSC at room temperature (RT) for 15
minutes each Wash twice: 0.5.times.SSC at 65.degree. C. for 20
minutes each High Stringency (Detects Sequences that Share at Least
80% Sequence Identity) Hybridization: 5.times.-6.times.SSC at
65.degree. C.-70.degree. C. for 16-20 hours Wash twice: 2.times.SSC
at RT for 5-20 minutes each Wash twice: 1.times.SSC at 55.degree.
C.-70.degree. C. for 30 minutes each Low Stringency (Detects
Sequences that Share at Least 50% Sequence Identity) Hybridization:
6.times.SSC at RT to 55.degree. C. for 16-20 hours Wash at least
twice: 2.times.-3.times.SSC at RT to 55.degree. C. for 20-30
minutes each.
[0171] F. Prion Proteins
[0172] This disclosure further provides compositions and methods
involving wild type and recombinant prion proteins. In some
embodiments, prion protein variants include the substitution of one
or several amino acids for amino acids having similar biochemical
properties (so-called conservative substitutions). Conservative
amino acid substitutions are likely to have minimal impact on the
activity of the resultant protein, such as it's ability to convert
PrP-sen to PrP-res. Further information about conservative
substitutions can be found, for instance, in Ben Bassat et al. (J.
Bacteriol., 169:751-757, 1987), O'Regan et al. (Gene, 77:237-251,
1989), Sahin-Toth et al. (Protein Sci., 3:240-247, 1994), Hochuli
et al. (Bio/Technology, 6:1321-1325, 1988) and in widely used
textbooks of genetics and molecular biology. In some examples,
prion protein variants can have no more than 3, 5, 10, 15, 20, 25,
30, 40, or 50 conservative amino acid changes. The following table
shows exemplary conservative amino acid substitutions that can be
made to a prion protein, for instance the recombinant prion
proteins shown in SEQ ID NOs: 1-7.
TABLE-US-00008 TABLE 2 Original Residue Conservative Substitutions
Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly
Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met
Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0173] G. Purification of Recombinant Prion Protein
[0174] To purify PrP-sen from recombinant (or natural) sources, the
composition is subjected to fractionation to remove various other
components from the composition. Various techniques suitable for
use in protein purification are well known. These include, for
example, precipitation with ammonium sulfate, PTA, PEG, antibodies
and the like, or by heat denaturation followed by centrifugation;
chromatography steps such as metal chelate, ion exchange, gel
filtration, reverse phase, hydroxylapatite, lectin affinity, and
other affinity chromatography steps; isoelectric focusing; gel
electrophoresis; and combinations of such and other techniques.
[0175] H. Sources of Samples for rPrP-Res Amplification Assays,
Such as rPrP-PMCA and QUIC Assays
[0176] The samples analyzed using the methods described herein can
include any composition capable of being contaminated with a prion.
Such compositions can include tissue samples or bodily fluids
including, but not limited to, blood, lymph nodes, brain, spinal
cord, tonsils, spleen, skin, muscles, appendix, olfactory
epithelium, cerebral spinal fluid, urine, feces, milk, intestines,
tears and/or saliva. Other compositions from which samples can be
taken for analysis, for instance, include food stuffs, drinking
water, forensic evidence, surgical implements, and/or mechanical
devices.
[0177] I. Methods for Detecting rPrP-res.sup.(Sc) in rPrP-res
Amplification Mixes, Such as rPrP-PMCA and QUIC Reaction Mixes
[0178] Once rPrP-res.sup.(Sc) has been generated using rPrP-res
amplification, such as using rPrP-PMCA (such as the QUIC assay),
rPrP-res.sup.(Sc) can be detected in the reaction mix. Direct and
indirect methods can be used for detection of rPrP-res.sup.(Sc) in
a reaction mix or serial reaction mix. For methods in which
rPrP-res.sup.(Sc) is directly detected, separation of newly-formed
rPrP-res.sup.(Sc) from remaining rPrP-sen usually is required. This
typically is accomplished based on the different natures of
rPrP-res.sup.(Sc) versus rPrP-sen. For instance, rPrP-res.sup.(Sc)
typically is highly insoluble and resistant to protease treatment.
Therefore, in the case of rPrP-res.sup.(Sc) and rPrP-sen,
separation can be by, for instance, protease treatment.
[0179] When rPrP-res.sup.(Sc) and rPrP-sen are separated by
protease treatment, reaction mixtures are incubated with, for
example, Proteinase K (PK). An exemplary protease treatment
includes digestion of the protein, for instance, rPrP-sen, in the
reaction mixture with 1-20 .mu.g/ml of PK for about 1 hour at
37.degree. C. Reactions with PK can be stopped prior to assessment
of prion levels by addition of PMSF or electrophoresis sample
buffer. Depending on the nature of the sample, incubation at
37.degree. C. with 1-50 .mu.g/ml of PK generally is sufficient to
remove rPrP-sen.)
[0180] rPrP-res.sup.(Sc) also can be separated from the rPrP-sen by
the use of ligands that specifically bind and precipitate the
misfolded form of the protein, including conformational antibodies,
certain nucleic acids, plasminogen, PTA and/or various peptide
fragments.
[0181] 1. Western Blot
[0182] In some examples, reaction mixtures fractioned or treated
with protease to remove rPrP-sen are then subjected to Western blot
for detection of rPrP-res.sup.(Sc) and the discrimination of
rHaPrP-res.sup.(Sc) from rHaPrP-res.sup.(spon). Typical Western
blot procedures begin with fractionating proteins by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions. The proteins are then electroblotted onto a
membrane, such as nitrocellulose or PVDF and probed, under
conditions effective to allow immune complex (antigen/antibody)
formation, with an anti-prion protein antibody. Exemplary
antibodies for detection of prion protein include the 3F4
monoclonal antibody, monoclonal antibody D13 (directed against
residues 96-106 (Peretz et al. (2001) Nature 412, 739-743)),
polyclonal antibodies R18 (directed against residues 142-154), and
R20 (directed against C-terminal residues 218-232) (Caughey et al.
(1991) J. Virol. 65, 6597-6603).
[0183] Following complex formation, the membrane is washed to
remove non-complexed material. An exemplary washing procedure
includes washing with a solution such as PBS/Tween, or borate
buffer. The immunoreactive bands are visualized by a variety of
assays known to those in the art. For example, the enhanced
chemoluminescence assay (Amersham, Piscataway, N.J.) can be
used.
[0184] If desired, prion protein concentration can be estimated by
Western blot followed by densitometric analysis, and comparison to
Western blots of samples for which the concentration of prion
protein is known. For example, this can be accomplished by scanning
data into a computer followed by analysis with quantitation
software. To obtain a reliable and robust quantification, several
different dilutions of the sample generally are analyzed in the
same gel.
[0185] 2. ELISA, Immunochromatographic Strip Assay, and
Conformation Dependent Immunoassay
[0186] As described above, immunoassays in their most simple and
direct sense are binding assays. Specific non-limiting immunoassays
of use include various types of enzyme linked immunosorbent assays
(ELISAs), immunochromatographic strip assays, radioimmunoassays
(RIA), and specifically conformation-dependent immunoassays.
[0187] In one exemplary ELISA, anti-PrP antibodies are immobilized
onto a selected surface exhibiting protein affinity, such as a well
in a polystyrene microtiter plate. Then, a reaction mixture
suspected of containing prion protein antigen is added to the
wells. After binding and washing to remove non-specifically bound
immune complexes, the bound prion protein can be detected.
Detection generally is achieved by the addition of another anti-PrP
antibody that is linked to a detectable label. This type of ELISA
is a simple "sandwich ELISA." Detection also can be achieved by the
addition of a second anti-PrP antibody, followed by the addition of
a third antibody that has binding affinity for the second antibody,
with the third antibody being linked to a detectable label.
[0188] In another exemplary ELISA, the reaction mixture suspected
of containing the prion protein antigen is immobilized onto the
well surface and then contacted with the anti-PrP antibodies. After
binding and washing to remove non-specifically bound immune
complexes, the bound anti-prion antibodies are detected. Where the
initial anti-PrP antibodies are linked to a detectable label, the
immune complexes can be detected directly. Again, the immune
complexes can be detected using a second antibody that has binding
affinity for the first anti-PrP antibody, with the second antibody
being linked to a detectable label.
[0189] Another ELISA in which protein of the reaction mix is
immobilized involves the use of antibody competition in the
detection. In this ELISA, labeled antibodies against prion protein
are added to the wells, allowed to bind, and detected by means of
their label. The amount of prion protein antigen in a given
reaction mix is then determined by mixing it with the labeled
antibodies against prion before or during incubation with coated
wells. The presence of prion protein in the sample acts to reduce
the amount of antibody against prion available for binding to the
well and thus reduces the ultimate signal. Thus, the amount of
prion in the sample can be quantified.
[0190] Irrespective of the format employed, ELISAs have certain
features in common, such as coating, incubating or binding, washing
to remove non-specifically bound species, and detecting the bound
immune complexes. These are described below.
[0191] In coating a plate with either antigen or antibody, one
generally incubates the wells of the plate with a solution of the
antigen or antibody, either overnight or for a specified period of
hours. The wells of the plate are then washed to remove
incompletely adsorbed material. Any remaining available surfaces of
the wells are then "coated" with a nonspecific protein that is
antigenically neutral with regard to the test antibodies. These
include bovine serum albumin, casein, and solutions of milk powder.
The coating allows for blocking of nonspecific adsorption sites on
the immobilizing surface, and thus reduces the background caused by
nonspecific binding of antibodies onto the surface.
[0192] It is customary to use a secondary or tertiary detection
means rather than a direct procedure with ELISAs, though this is
not always the case. Thus, after binding of a protein or antibody
to the well, coating with a non-reactive material to reduce
background, and washing to remove unbound material, the
immobilizing surface is contacted with the biological sample to be
tested under conditions effective to allow immune complex
(antigen/antibody) formation. Detection of the immune complex then
requires a labeled secondary binding ligand or antibody, or a
secondary binding ligand or antibody in conjunction with a labeled
tertiary antibody or third binding ligand.
[0193] "Under conditions effective to allow immune complex
(antigen/antibody) formation" means that the conditions preferably
include diluting the antigens and antibodies with solutions such as
BSA, bovine gamma globulin, milk proteins, and phosphate buffered
saline (PBS)/Tween. These added agents also tend to assist in the
reduction of nonspecific background. "Suitable" conditions also
mean that the incubation is at a temperature and for a period of
time sufficient to allow effective binding. Incubation steps are
typically from about 1 to 2 to 4 hours, at temperatures preferably
on the order of 25.degree. C. to 27.degree. C., or can be overnight
at about 4.degree. C. or so.
[0194] Following all incubation steps in an ELISA, the contacted
surface is washed so as to remove non-complexed material. An
exemplary washing procedure includes washing with a solution such
as PBS/Tween or borate buffer. Following the formation of specific
immune complexes between the test sample and the originally bound
material, and subsequent washing, the occurrence of even minute
amounts of immune complexes can be determined.
[0195] To provide a detecting means, the second or third antibody
generally will have an associated label to allow detection. In some
examples, this is an enzyme that will generate color development
upon incubating with an appropriate chromogenic substrate. Thus,
for example, the first or second immune complex is contacted and
incubated with a urease, glucose oxidase, alkaline phosphatase or
hydrogen peroxidase-conjugated antibody for a period of time and
under conditions that favor the development of further immune
complex formation (for instance, incubation for two hours at room
temperature in a PBS-containing solution such as PBS-Tween).
[0196] After incubation with the labeled antibody, and subsequent
to washing to remove unbound material, the amount of label is
quantified, for instance, by incubation with a chromogenic
substrate such as urea and bromocresol purple or
2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantification is then achieved by measuring the degree of color
generation, for instance, using a visible spectra
spectrophotometer.
[0197] J. rPrP-sen Labeling
[0198] In certain embodiments, the recombinant PrP-sen substrate
protein can be labeled to enable high sensitivity of detection of
protein that is converted into rPrP-res.sup.(Sc). For example,
rPrP-sen can be radioactively labeled, epitope tagged, or
fluorescently labeled. The label can be detected directly or
indirectly. Radioactive labels include, but are not limited to
.sup.125I, .sup.32P, .sup.33P, and .sup.35S.
[0199] The mixture containing the labeled protein is subjected to
an rPrP-Res amplification assay, such as rPrP-PMCA or QUIC, and the
product detected with high sensitivity by following conversion of
the labeled protein after removal of the unconverted protein for
example by proteolysis. Alternatively, the protein can be labeled
in such a way that a signal can be detected upon the conformational
changes induced during conversion. An example of this is the use of
FRET technology, in which the protein is labeled by two appropriate
fluorophores, which upon refolding become close enough to exchange
fluorescence energy (see for example U.S. Pat. No. 6,855,503).
[0200] In certain embodiments, cysteine residues are placed at
positions 94 and 95 of the hamster prion protein sequence in order
to be able to selectively label the rPrP-sen at those sites using
sulfhydryl-reactive labels, such as pyrene and fluorescein linked
to maleimide-based functional groups. In certain embodiments, these
tags do not interfere with conversion but allow much more rapid,
fluorescence-based detection of the rPrP-PMCA reaction product. In
one example, pyrenes in adjacent molecules of rPrP-res are held in
close enough proximity to allow eximer formation, which shifts the
fluorescence emission spectrum in a distinct and detectable manner
Free pyrenes released from, or on, unconverted rPrP-sen molecules
are unlikely to form eximer pairs. Thus, the rPrP-Res amplification
reaction can be run in a multiwell plate, digested with proteinase
K, and then eximer fluorescence can be measured to rapidly test for
the presence of rPrP-res.sup.(Sc). Sites 94 and 95 are chosen for
the labels because the PK-resistance in this region of PrP-res
distinguishes rPrP-res.sup.(Sc) from rPrP-res.sup.(spon), giving
rise to the 17 kDa rPrP-res band. Other positions in the
PK-resistant region(s) that distinguish the 17-kDa
rHaPrP-res.sup.(Sc) fragment from all rHaPrP-res.sup.(spon)
fragments also can work for this purpose.
[0201] In certain other embodiments, the use of a
fluorescently-tagged rPrP-sen substrate for the reaction is
combined with the use an immunochromatographic strip test with an
immobilized rPrP-res specific antibody (for example, from Prionics
AG, Schlieren-Zurich, Switzerland). Binding of the rPrP-res to the
antibody is then detected with a fluorescence detector.
[0202] K. Antibody Generation
[0203] In certain embodiments, the present disclosure involves
antibodies, such as antibodies that recognize PrP proteins. For
example, antibodies are used in many of the methods for detecting
prions (for instance, Western blot and ELISA). In addition to
antibodies generated against full length proteins, antibodies also
can be generated in response to smaller constructs comprising
epitopic core regions, including wild-type and mutant epitopes.
[0204] As used herein, the term "antibody" is intended to refer
broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD
and IgE. Generally, IgG and/or IgM are used because they are the
most common antibodies in the physiological situation and because
they are most easily made in a laboratory setting. Monoclonal
antibodies are recognized to have certain advantages, for instance
reproducibility and large-scale production. The monoclonal
antibodies can be of human, murine, monkey, rat, hamster, rabbit
and even chicken origin.
[0205] The term "antibody" is used to refer to any antibody-like
molecule that has an antigen binding region, and includes antibody
fragments such as Fab', Fab, F(ab')2, single domain antibodies
(DABs), Fv, scFv (single chain Fv), and the like. The techniques
for preparing and using various antibody-based constructs and
fragments are well known. Means for preparing and characterizing
antibodies are also well known.
[0206] mAbs can be prepared through use of well-known techniques,
such as those exemplified in U.S. Pat. No. 4,196,265. mAbs can be
further purified, if desired, using filtration, centrifugation and
various chromatographic methods such as HPLC or affinity
chromatography. Fragments of the monoclonal antibodies of the
disclosure can be obtained from the monoclonal antibodies so
produced by methods which include digestion with enzymes, such as
pepsin or papain, and/or by cleavage of disulfide bonds by chemical
reduction. Alternatively, monoclonal antibody fragments encompassed
by the present disclosure can be synthesized using an automated
peptide synthesizer.
It also is contemplated that a molecular cloning approach can be
used to generate mAbs.
[0207] L. Screening for Modulators of Prion Function
[0208] The disclosed assay also can be used to identify compounds
that modify the ability of prions to replicate, such as compounds
that would be candidates for the treatment of prion diseases. Thus,
the method for screening compounds includes performing an rPrP-Res
amplification assay on control reaction mixtures, and reaction
mixtures including the test compound are accessed for levels of
rPrP-res.sup.(Sc) following amplification. When a difference
between the levels of rPrP-res.sup.(Sc) in the test versus control
reaction mixtures is detected, compounds could be identified that
either enhance or inhibit conversion of rPrP-sen to
rPrP-res.sup.(Sc). These assays can include random screening of
large libraries of candidate substances; alternatively, the assays
can be used to focus on particular classes of compounds selected
with an eye towards structural attributes that are believed to make
them more likely to modulate the function of prions.
[0209] By function, it is meant that one can determine the
efficiency of conversion by assaying conversion of a standard
amount of rPrP-sen into rPrP-res.sup.(Sc) by a known amount of
prion. This can be determined by, for instance, quantitating the
amount of rPrP-res.sup.(Sc) in a reaction mix following a certain
number of cycles of rPrP-PMCA or QUIC.
[0210] As used herein, the term "candidate substance" refers to any
molecule that potentially can inhibit or enhance prion function
activity. The candidate substance can be a protein or fragment
thereof, a small molecule, a polymer or even a nucleic acid
molecule. The most useful pharmacological compounds can be
compounds that are structurally related to prion protein or prion
protein ligands. Using lead compounds to help develop improved
compounds is known as "rational drug design," and includes not only
comparisons with known inhibitors and activators, but predictions
relating to the structure of target molecules. The goal of rational
drug design is to produce structural analogs of biologically active
polypeptides or target compounds. By creating such analogs, it is
possible to fashion drugs that are more active or stable than the
natural molecules, and that have different susceptibility to
alteration or which can affect the function of various other
molecules. In one approach, one generates a three-dimensional
structure for a target molecule, or a fragment thereof, for
instance by x-ray crystallography, computer modeling, or by a
combination of both approaches.
[0211] It also is possible to use antibodies to ascertain the
structure of a target compound activator or inhibitor. In
principle, this approach yields a pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein
crystallography altogether by generating anti-idiotypic antibodies
to a functional, pharmacologically active antibody. As a mirror
image of a mirror image, the binding site of an anti-idiotype would
be expected to be an analog of the original antigen. The
anti-idiotype could then be used to identify and isolate peptides
from banks of chemically- or biologically-produced peptides.
Selected peptides would then serve as the pharmacore.
Anti-idiotypes can be generated using the methods described herein
for producing antibodies, using an antibody as the antigen.
[0212] Alternatively, small molecule libraries can be acquired that
are believed to meet the basic criteria for useful drugs in an
effort to identify useful compounds by large-scale screening.
Screening of such libraries, including combinatorially generated
libraries (for instance, peptide libraries), is a rapid and
efficient way to screen large number of related (and unrelated)
compounds for activity. Combinatorial approaches also lend
themselves to rapid evolution of potential drugs by the creation of
second, third and fourth generation compounds modeled on active,
but otherwise undesirable compounds.
[0213] Candidate compounds can include fragments or parts of
naturally-occurring compounds, or can be found as active
combinations of known compounds, which are otherwise inactive.
Compounds isolated from natural sources, such as animals, bacteria,
fungi, plant sources, including leaves and bark, and marine
samples, can be assayed as candidates for the presence of
potentially useful pharmaceutical agents. It will be understood
that the pharmaceutical agents to be screened also could be derived
or synthesized from chemical compositions or man-made compounds.
Thus, it is understood that the candidate substance identified by
the present disclosure can be peptide, polypeptide, polynucleotide,
glycans, synthetic polymers, small molecule inhibitors or any other
compound(s) that can be designed through rational drug design
starting from known inhibitors or stimulators. Other suitable
modulators include antibodies (including single chain antibodies),
each of which would be specific for the target molecule. Such
compounds are described in greater detail above.
[0214] In addition to the modulating compounds initially
identified, other sterically similar compounds can be formulated to
mimic the key portions of the structure of the modulators. Such
compounds, which can include peptidomimetics of peptide modulators,
can be used in the same manner as the initial modulators. Preferred
modulators of prion replication would have the ability to cross the
blood-brain barrier since a large number of prion manifest
themselves in the central nervous system.
[0215] An inhibitor can be one that exerts its activity directly on
the PrP-res, on the PrP-sen, or on factors required for the
conversion of PrP-sen to PrP-res. Regardless of the type of
inhibitor or activator identified by the present screening methods,
the effect of the inhibition or activation by such a compound
results in altered prion amplification or replication as compared
to that observed in the absence of the added candidate
substance.
[0216] M. Kits
[0217] Any of the compositions described herein can be included in
a kit for carrying out rPrP-PCMA or QUIC. In a non-limiting
example, recombinant PrP-sen, prion conversion factors,
decontamination solution, and/or conversion buffer with a metal
chelator are provided in a kit. The kit further can include
reagents for expressing or purifying rPrP-sen. The kit also can
include pre-labeled rPrP-sen or reagents that can be used to label
the rPrP-sen, with for example, radio isotopes or fluorophores.
[0218] In some embodiments, kits are provided for amplification and
detection of prion in a sample. In these embodiments, a kit can
include, in suitable container, one or more of the following: 1) a
conversion buffer; 2) decontamination solution; 3) a positive
control, prion containing sample; 4) a negative control sample, not
containing prion; or 5) reagents for detection of rPrP-res.
[0219] Regents for the detection of prions can include one or more
of the following: pre coated microtiter plates for ELISA and/or CDI
detection of rPrP-res; or antibodies for use in ELSA, CDI, strip
immunochromatography or Western blot detection methods.
[0220] Additionally, kits of the disclosure can contain one or more
of the following: protease free water; copper salts for inhibiting
rPrP-res amplification; EDTA solutions for enhancing prion
replication; Proteinase K for the separation of rPrP-res from
rPrP-sen; fractionation buffers for the separation of rPrP-res from
rPrP-sen, modified, or labeled proteins (to increase sensitivity of
detection); or conversion factors (to enhance efficiency of
amplification).
[0221] In certain embodiments, the conversion buffer is supplied in
a "ready for amplification format" where it is allocated in a
microtiter plate such that the sample and rPrP-sen can be added to
first well, and subjected to primary reaction and amplification.
Thereafter a portion of the reaction mix is moved to an adjacent
well with additional rPrP-sen added for serial rPrP-res
amplification. These steps can be repeated across the microtiter
plate for multiple serial amplifications.
[0222] The components of the kits can be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, plate, flask,
bottle, syringe or other container, into which a component can be
placed, and optionally, suitably aliquoted. Where there is more
than one component in the kit (labeling reagent and label can be
packaged together), the kit also generally will contain a second,
third or other additional container into which the additional
components can be separately placed. However, various combinations
of components can be included in a vial. The kits also typically
will include a means for containing proteins, and any other reagent
containers in close confinement for commercial sale. Such
containers can include injection or blow-molded plastic containers
into which the desired vials are retained.
[0223] When components of the kit are provided in one and/or more
liquid solutions, the liquid solution is typically an aqueous
solution that is sterile and proteinase free. In some cases
protein-based compositions are lyophilized to prevent degradation
and/or the kit or components thereof can be stored at a low
temperature (for instance, less than about 4.degree. C.). When
reagents and/or components are provided as a dry powder and/or
tablets, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent also can be
provided in another container means.
[0224] N. rPrP-Res Amplification Assays (rPrP-PMCA/QUIC Assays)
Using Samples from Humans, Bovines, and Other Species
[0225] As described above, it is desirable to carry out rPrP-PMCA
and/or QUIC assays in a variety of species. For instance, the
assays are useful in screening bovine, sheep, and cervid
individuals or populations for prion diseases, for example to
ensure the safety of the food supply. In some instances,
rPrP-PMCA/QUIC assays are useful for diagnosing prion disease in a
human or veterinary subject.
[0226] The rPrP-sen chosen may be chosen from the same species as
the test sample, or it may be of a different species. For example,
a hamster or mouse rPrP-sen can be used to amplify a human or sheep
test sample. In particular examples hamster rPrP (rHaPrP) is used
because it is particularly effective in amplification reactions,
(such as QUIC) not only of hamster PrP-res but of PrP-res from
humans and sheep as well. For those embodiments in which the
rPrP-sen is from the same species as the test sample, it is also
desirable to create rPrP-sen from a variety of species that may be
tested. As described above in greater detail, in general, the
partial prion protein sequence expressed as rPrP-sen corresponds to
the polypeptide sequences of the natural mature full-length PrP c
molecule, meaning that the rPrP-sen polypeptide lacks both the
amino-terminal signal sequence and carboxy-terminal
glycophosphatidylinositol-anchor attachment sequence. Thus, in some
embodiments, a hamster rPrP includes amino acids 23-231 (SEQ ID NO:
1) of hamster prion protein sequence (SEQ ID NO: 8), a bovine
rPrP-sen includes amino acids 25-241 (SEQ ID NO: 5) of a bovine
prion protein sequence, whereas a human rPrP-sen includes amino
acids 23-231 (SEQ ID NOs: 3, 4) of a human prion protein sequence
(SEQ ID NOs: 10, 11), an ovine rPrP-sen includes amino acids 25-233
(SEQ ID NO: 6) of an ovine prion protein sequence, and a cervid
rPrP-sen includes amino acid residues 25-234 (SEQ ID NO: 7) of a
cervid prion protein sequence or residues 90-231 of the hamster
sequence (SEQ ID NO: 8). However, the rPrP protein is not limited
to these particular portions of each sequence. Other exemplary
portions are disclosed above.
[0227] As described above in greater detail, the test sample can be
any tissue sample from a human or veterinary subject, for instance
a brain sample, peripheral organ sample (such as blood, tonsil,
spleen, or another lymphoid organ), various excretia or a CSF
sample. In the case of living subjects, blood, excretia, or CSF
samples are easily obtained with relatively non-invasive
techniques. In the case of samples from deceased subjects, brain or
other tissue samples are easily obtained.
[0228] Once the rPrP-sen and test samples have been obtained, an
rPrP-PMCA or QUIC assay is performed as described in detail above.
Results generally are available within 24-72 hours, which greatly
speeds diagnosis, treatment, and/or disease
containment/decontamination efforts.
[0229] The following Examples are provided to illustrate certain
particular features and/or embodiments. These Examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1: Materials and Methods
[0230] This example describes materials and methods used to carry
out Examples 2-8. Although particular methods are described, it is
understood that other methods can be used.
Recombinant Prion Protein Expression and Purification
[0231] DNA sequences coding for hamster (GENBANK.RTM. Accession No.
M14054) and mouse (GENBANK.RTM. Accession No. BC006703) prion
protein residues 23-230 or 90-230 were amplified by standard PCR,
ligated into the Kanamycin selective pET41 vector (EMD Biosciences)
as NdeI/HindIII inserts, and their sequences were verified. After
transforming the plasmids into E. coli Rosetta cells (EMD
Biosciences), the rPrP-sen was expressed using the Overnight
Express Autoinduction system according to the instructions from the
manufacturer (EMD Biosciences). A typical mass of wet cell paste
was 8-9 grams per Liter of Luria-Bertani media. Cell pellets were
lysed with BugBuster.TM. and Lysonase.TM. (EMD Bioscieces).
Approximately 25 mL of BugBuster.TM. with 50 .mu.L of lysonase and
one Complete EDTA-free protease inhibitor tablet (Roche) was used
for each gram of harvested bacterial cells. This lysis mixture was
stirred with the bacterial cells in an ice bath. This mixture was
then subjected to periodic sonication (15 pulses of 15 seconds over
30 minutes at full power) to facilitate lysis. Inclusion bodies
containing rPrP-sen were isolated by centrifugation and then were
twice washed with 0.1.times. BugBuster.TM. and pelleted by
centrifugation in 50 mL centrifuge tubes. The enriched rPrP was
further purified by modifications to the method of Zahn et al.,
(1997) FEBS Lett. 417, 400-404. The washed inclusion bodies were
then suspended in aqueous 8 M Guanidine hydrochloride and this
mixture was pelleted by centrifugation to remove cell debris. The
supernatant containing denatured rPrP-sen was stirred with Ni-NTA
Superflow (Qiagen) resin and then loaded onto an XK16/20 column (GE
Healthcare) and then washed with denaturing buffer (6 M Guanidine
hydrochloride, 100 mM sodium phosphate, 10 mM Tris, pH 8.0) and
refolded with a linear gradient over 6 hours at a flow rate of 0.75
to 1 mL/minute using an AKTA Explorer 10. The protein was then
eluted with 100 mM sodium phosphate (pH 5.8), 500 mM imidazole, 10
mM Tris. Pooled fractions were diluted to 0.2 mg/ml with water,
filtered and dialyzed against 10 mM phosphate. The 10 mM phosphate
dialysis buffer was diluted from a 1 M stock of the same buffer (pH
5.8) immediately prior to dialysis. After dialysis against a total
of 4 L (2.times.2 L) at 4.degree. C., with the second treatment
overnight, the protein solution was sterile filtered through a 0.22
.mu.m 150 mL filter unit (Millipore) and the protein concentration
of rPrP was determined by the method of Bradford or by A.sub.280
nm. Purity of the final protein preparations was estimated at
.gtoreq.99% when analyzed by SDS-PAGE, immunoblotting and MALDI
mass spectrometry.
[0232] Differences of this method to that of Zahn et al. include
the isolation of inclusion bodies that were not isolated in Zahn et
al., lysis in non-denaturing Bug Buster instead of lysis in a
denaturing buffer, the elimination of the use of a glutathione
denaturing buffer, and the elimination of the histidine tag.
rPrP-PMCA
[0233] rPrP-PMCA reactions were prepared in 0.2 ml PCR tubes as 80
.mu.l solutions containing PBS, pH 7.4, containing 0.05% (w/v) SDS
and 0.05% TRITON.RTM. X-100 (TX-100), except as shown in FIG. 1,
where 0.1% of each detergent was used. rHaPrP-sen was present at
0.1 mg/ml (4 .mu.M). The reactions were seeded with brain
homogenate from Syrian golden hamsters affected with the 263K
scrapie strain (ScBH) or purified PrP.sup.Sc (HaPrP.sup.Sc) from
the same source (Raymond & Chabry in Techniques in Prion
Research (eds. Lehmann & Grassi) 16-26 (Birkhauser Verlag,
Basel, 2004)). The PrP.sup.Sc concentration in the ScBH was
estimated by semiquantitative immunoblotting against purified
HaPrP.sup.Sc standards. Reactions were immersed in water at
37.degree. C. and subjected to repeated cycles of sonication
(Misonix Model 3000), based on previous methods (see, for instance,
Saa et al., (2006) J. Biol. Chem. 281, 35245-35252) with minor
modifications. In brief, sonication was performed over 24 hours
(constituting one round) with 40 second pulses every 60 minutes at
maximum power. Unsonicated controls were incubated at 37.degree.
C.
[0234] Although rPrP-PMCA will work under a variety of conditions,
the optimal conditions that supported specific PrP.sup.Sc-seeded
rPrP-PMCA include the combination of about 0.05-0.1% of an anionic
detergent such as SDS and about 0.05-0.1% of a nonionic detergent
such as TX-100. These conditions are particularly effective at
promoting the formation of rHaPrP-res.sup.(Sc) (notably the 17 kDa
PK-resistant species), while reducing rHaPrP-res.sup.(spon)
formation within the first 24 hours of unseeded reactions. Previous
studies showed that prion protein aggregation can be prompted by
low concentrations of anionic detergents (Xiong et al., (2001) J.
Neurochem. 79, 669-678). Other conditions can promote the
spontaneous formation of rPrP-res that includes a .about.17-kDa
fragment (Bocharova et al. (2006) J. Biol. Chem. 281, 2373-2379),
so seeding with PrP.sup.Sc is not always required for the formation
of rPrP-res with a banding pattern like that of
rHaPrP-res.sup.(Sc). However, under the rPrP-PMCA conditions set
forth in this example, PrP.sup.Sc seeding is required, allowing for
clear and consistent discrimination between HaPrP.sup.Sc-seeded and
unseeded reactions. Without being bound by theory, it is believed
that these specific detergent conditions can partially unfold
rPrP-sen, allowing productive contacts between PrP.sup.Sc and
rPrP-sen that would not otherwise occur spontaneously between
rPrP-sen molecules.
QUIC
[0235] A different method from PMCA uses Quaking Induced Conversion
(QUIC), in which shaking of the reaction mixture replaces
sonication for disaggregating aggregates formed during cyclic
amplification. Of course both shaking and sonication can be used in
an amplification reaction, for example in alternating cycles. In
the particular examples of QUIC disclosed herein only shaking of
reaction vessels is used.
[0236] Either purified PrP.sup.Sc or scrapie brain homogenate were
used to seed the conversion of rPrP-sen to protease-resistant forms
in reactions performed in 0.1% sodium dodecyl sulfate and 0.1%
TRITON.RTM. X-100, in PBS at 37.degree. C. in 0.5 ml tubes. Tube
shaking was done at 1500 rpm in an EPPENDORF THERMOMIXER.RTM. R.
Proteinase K digestions and immunoblotting were performed as
described in the step-by-step protocol, below.
[0237] For comparing PK-resistant QUIC reaction products, 24-hour
unshaken reactions and reactions were shaken with or without 0.1 mm
glass cell disruption beads (Scientific Industries). These
reactions were seeded with 10 ng of purified hamster PrP.sup.Sc
with 0.2 mg/ml hamster rPrP-sen and a 50 .mu.l reaction volume. The
tubes were subjected to cycles of 2 minutes of shaking and 28
minutes without shaking. C-terminal antibody R20 was used for the
immunoblot.
[0238] For 20-hour QUIC reactions performed with the varying
rPrP-sen concentrations, reaction volumes, and seed amounts, the
seed amounts approximate the estimated quantity of PrP.sup.Sc added
in 2-.mu.l aliquots dilutions of scrapie brain homogenate (in 1%
normal brain homogenate). The tubes were subjected to cycles of 10
seconds of shaking and 110 seconds without shaking. R20 was used
for the immunoblot.
For extended reactions to QUIC sensitivity to small amounts of
scrapie brain homogenate seed, 65-hour and 95-hour QUIC reactions
were carried out as described above, and 0.2 mg/ml rPrP-sen, were
used for 100-.mu.l reaction volumes. Scrapie brain homogenate seed
dilutions containing the designated amount of PrP.sup.Sc, were
subjected to cycles of 10 seconds shaking and 110 seconds without
shaking.
[0239] In other examples, 48-hour reaction times were used with
reduced detergent concentrations (0.05% SDS and 0.05% TRITON.RTM.
X-100). For the second round, 10% of the volume of the first round
reaction products were diluted into 9 volumes of reaction buffer
containing fresh rPrP-sen. PK-digestions and immunoblotting using
either R20 or D13 primary antibodies were performed as described
below.
[0240] For seeding with CSF samples, aliquots (2 .mu.l) of CSF
taken from normal hamsters (n=3) or hamsters in the clinical phase
of scrapie (n=6) were used to seed QUIC reactions using the
conditions, and immunoblots were carried out using the PK-digested
products of the first 48-hour round. Ten percent of each first
round reaction volume was used to seed a second 48-hour round of
QUIC. Antibodies R20 and D13 were used for the immunoblots.
CSF Collection
[0241] Hamsters were heavily sedated with isofluorane and
exsanguinated using cardiac puncture. Skin and muscles at the back
of the neck were dissected away avoiding blood vessels and
meninges. A small hole was made at the medial aperture in the
meninges using a 263/4 G needle and a Drummond micropipette was
quickly inserted into the hole. CSF filled the micropipette by
capillary action. Rocky Mountain Laboratories is an
AALAC-accredited facility, and all animal procedures were approved
by the institution's Animal Use and Care Committee.
Proteinase K Digestion, SDS-PAGE and Western Blotting
[0242] At the end of the reaction, 5 .mu.l of the reaction sample
(1 .mu.g of rPrP) was diluted five-fold in PBS with 0.1% SDS and
digested with the specified PK:rHaPrP ratio (0.025:1=1 .mu.g/ml of
PK, 0.25:1=10 .mu.g/ml of PK, or 0.5:1=20 .mu.g/ml of PK) for 1
hour at 37.degree. C. PEFABLOC.RTM.
(4-(2-Aminoethyl)-benzensulfonyl fluoride (Roche) was then added to
a final concentration of 4 mM. For those samples analyzed by
western blotting, 20 .mu.g of thyroglobulin was added and the
protein was precipitated with 4 volumes of methanol and stored at
-20.degree. C. prior to centrifugation and aspiration of the
methanol. Pellets were suspended in sample buffer (4 M urea, 4%
SDS, 2% 0-mercaptoethanol, 8% glycerol, 0.02% bromophenol blue and
50 mM Tris-HCl pH6.8), subjected to SDS-PAGE using 10% BisTris
NUGPAGE.RTM. (polyacrylamide) gels (Invitrogen), and transferred to
IMMOBILON.TM. P membrane (Millipore). The membrane was probed with
D13 (Peretz et al. (2001) Nature 412, 739-743), R20 (Caughey et
al., (1991) J. Virol. 65, 6597-6603), or R18 antibodies at 1:10,000
dilutions as specified, and visualized by ATTOPHOS.RTM. AP
Fluorescent Substrate System
(2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate [BBTP])
(Promega) according to the manufacturer's recommendations. For
silver staining, methanol precipitation was omitted and the
PK-digested samples were mixed with 5.times. sample buffer, boiled,
and analyzed by SDS-PAGE.
Electron Microscopy
[0243] rHaPrP-res.sup.(spon) and rHaPrP-res.sup.(Sc) from fourth
round reactions were treated with PK (PK:PrP ratio of 0.025:1) at
37.degree. C. for one hour, after which 5 mM PEFABLOC.RTM.
(4-(2-Aminoethyl)-benzensulfonyl fluoride) was added. These and PK
untreated samples were pelleted by centrifugation for 30 minutes at
16,100 g, washed twice with PBS or water, and sonicated for one
minute. The samples were then settled onto Formvar-coated grids for
15 minutes, washed three times with sterile water, and stained with
methylamine tungstate for one minute. Excess stain was removed by
filter paper and the samples were dried at room temperature. Images
were obtained with an 80 kV in a Hitachi H-7500 electron microscope
and an AMT XR-100 digital camera system (Advanced Microscopy
Techniques, Danvers, Mass.).
Spectral Analysis
[0244] rHaPrP-res.sup.(spon) and rHaPrP-res.sup.(Sc) (seeded with
purified HaPrPSc) from third round reactions were pelleted by
centrifugation for 30 minutes at 16,100 g and twice washed in 10
.mu.l of sterile water. Slurried pellets were applied to a Golden
Gate Single Reflection Diamond Attenuated Total Reflectance unit
purged with dehydrated air and dried under a stream of nitrogen.
Data collection was performed using a System 2000 IR instrument
(Perkin-Elmer) with a liquid nitrogen cooled nbl MCT detector and
the following parameters: 20.degree. C., 1 cm.sup.-1 resolution, 5
cm/s optical path difference velocity, 500 scans 1800-1400
cm.sup.-1 scan range, and 0.5 cm.sup.-1 data interval. Primary
spectra were obtained by subtracting the corresponding buffer and
water vapor spectra. Fourier-self deconvoluted spectra were
calculated from the primary difference spectra using a gamma of
19.5 and a smoothing length of 95%. The software used for spectral
analyses was Spectrum v2.00 (Perkin-Elmer).
Example 2: Spontaneous Conversion of rPrP-sen
[0245] This Example describes the identification of an exemplary
set of reaction conditions that allow clear discrimination between
PrP.sup.Sc-seeded and unseeded reaction products. Although
particular reaction conditions are specified, one will recognize
that other reaction conditions can be used.
[0246] Development of a PMCA-like reaction for PrP.sup.Sc
amplification using rPrP-sen as a substrate requires conditions
that allow for clear discrimination between PrP.sup.Sc-seeded and
unseeded reaction products. Initial trials revealed that in 0.1%
SDS with periodic sonications, bacterially expressed recombinant
mouse PrP-sen (rMoPrP-sen; FIG. 5) and hamster PrP-sen (rHaPrP-sen)
converted spontaneously to thioflavin T-positive, proteinase K
(PK)-resistant forms designated rMoPrP-res.sup.(spon) and
rHaPrP-res.sup.(spon), respectively. The fragments generated by
PK-digestion of rMoPrP-res.sup.(spon) and rHaPrP-res.sup.(spon)
were 10-12 kDa, that is, much smaller than the .about.17-19 kDa
fragment typical of unglycosylated scrapie PrP.sup.Sc and
PrP.sup.Sc-induced rPrP-res.sup.8-10. When seeded into fresh
solutions of rMoPrP-sen and rHaPrP-sen, respectively,
rMoPrP-res.sup.(spon) and rHaPrP-res.sup.(spon) elicited more
thioflavin T-positive rPrP-res.sup.(spon), even without sonication
(FIG. 6). However, the addition of 0.1% TX-100 to the 0.1% SDS
permitted seeded rPrP-res.sup.(spon) accumulation, but often
delayed its spontaneous formation for >24 hours even in
sonicated reactions. Thus, these conditions were selected for
subsequent attempts to seed rHaPrP-sen conversion with
PrP.sup.Sc.
Example 3: Seeding of rPrP-sen Conversion by PrP.sup.Sc
[0247] This example demonstrates that scrapie PrP.sup.Sc can seed
the conversion of rPrP-sen to rPrP-res.
[0248] Scrapie PrP.sup.Sc purified from hamster brains
(HaPrP.sup.Sc; Raymond & Chabry in Techniques in Prion Research
(eds. Lehmann & Grassi) 16-26 (Birkhauser Verlag, Basel, 2004))
was used to seed the conversion of rHaPrP-sen. PK-resistant
fragments seeded by PrP.sup.Sc (rHaPrP-res.sup.(Sc), where (Sc)
refers to seeding by PrP.sup.Sc) were generated with
seed-to-substrate ratios of 1:100 (400 ng HaPrP.sup.Sc) and 1:1,000
(40 ng HaPrP.sup.Sc) in both the unsonicated and sonicated
reactions, but, when sonicated were much more abundant and less
dependent on the amount of seed (FIG. 1A). When analyzed by
immunoblotting using an anti-PrP antibody R20 directed toward
C-terminal residues 219-232, rHaPrP-res.sup.(Sc) consisted of 4
PK-resistant fragments (11, 12, 13 and 17 kDa). In contrast, and as
expected, the unseeded reactions gave either no PK-resistant bands
(FIG. 1A) or, more rarely, rHaPrP-res.sup.(spon) with only the
smaller 10-, 11- and 12-kDa fragments (FIG. 1A). The 17-kDa
rHaPrP-res.sup.(Sc) band also was not observed in the absence of
rHaPrP-sen substrate, demonstrating that the HaPrP.sup.Sc seed
itself did not display this band (FIG. 1A). Collectively, these
data demonstrate that HaPrP.sup.Sc-seeded rPrP-sen conversion
reactions can be distinguished from unseeded reactions by
immunoblot comparison of the PK-resistant banding patterns. Most
notable was the formation of the 17 kDa-band in the
HaPrP.sup.Sc-seeded reactions as has been observed previously in
substoichiometric conversion reactions with rPrP-sen (Iniguez et
al., (2000) J. Gen. Virol. 81, 2565-2571; Kirby et al., (2003) J.
Gen. Virol. 84, 1013-1020; Eiden et al., (2006) J. Gen. Virol. 87,
3753-3761).
[0249] The ability of rHaPrP-res.sup.(Sc) to seed additional rounds
of rHaPrP-res.sup.(Sc) amplification was tested by diluting
products of the first-round HaPrP.sup.Sc-seeded reaction seeded
(FIG. 1A) into fresh rHaPrP-sen substrate. For brevity, the term
"rPrP-PMCA" is used when referring to the use of rPrP-sen as a
substrate in combination with periodic sonication and (optionally)
cyclic dilutions of reaction products into fresh substrate to
detect PrP.sup.Sc and amplify rHaPrP-res.sup.(Sc). Without
sonication, the rHaPrP-res.sup.(Sc) produced in both the first and
second rounds decreased with dilution of the seed (FIGS. 1A, 1B).
With sonication, the yield was less dependent upon seed
concentration, with similarly high levels of rHaPrP-res.sup.(Sc)
produced at each dilution (FIGS. 1A, 1B). Similar levels of
rHaPrP-res.sup.(Sc) were produced in each of five consecutive
rounds of amplification with the products of each round diluted
1,000-fold into newly prepared rHaPrP-sen. Overall, periodic
sonication reduced the amount of HaPrP.sup.Sc required to initiate
robust rHaPrP-res.sup.(Sc) generation.
[0250] To further clarify the difference in the PK susceptibility
between rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon), immunoblots
were performed with additional antibodies (FIG. 1C). Monoclonal
antibody D13 (directed against residues 96-106 (Peretz et al.
(2001) Nature 412, 739-743)) specifically recognized the
PrP.sup.Sc-induced 17 kDa band but not the lower molecular weight
fragments. In contrast, the polyclonal antibody R18 (directed
against residues 142-154 (Peretz et al. (2001) Nature 412,
739-743)) recognized 17 kDa, 13 kDa and 12 kDa fragments in
rHaPrP-res.sup.(Sc) and 12 kDa fragments in rHaPrP-res.sup.(spon).
The C-terminal antibody R20 reacted with all of the rHaPrP-res
fragments, including the shortest 10 kDa fragment that appears to
be specific for rHaPrP-res.sup.(spon), indicating these fragments
differed primarily at their N-termini. Distinct fragment patterns
were observed for rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon)
over a wide range of PK:rPrP ratios (FIG. 1C) and detergent
compositions (FIG. 7). In agreement with the R20 immunoblots (FIG.
1C), silver-stained SDS-PAGE gels of PK-digested third-round
reaction products confirmed that rHaPrP-res.sup.(Sc) comprised
primarily the 11, 12, 13 and 17 kDa bands while
rHaPrP-res.sup.(spon) comprised the 10, 11 and 12 kDa bands (FIG.
1D). Thus, PrP.sup.Sc-seeded and non-seeded reaction products
differed in their susceptibility to proteolytic cleavage, providing
compelling evidence for fundamental differences in
conformation.
Example 4: Ultrasensitive Detection of PrP.sup.Sc
[0251] To determine the minimum amount of PrP.sup.Sc detectable by
rPrP-PMCA, scrapie brain homogenates (ScBH) were diluted serially
with 1% normal brain homogenate (NBH) and were used to seed
rPrP-PMCA reactions. The PK-treated products were analyzed by
immunoblotting with D13 antibody. After a single round, the 17-kDa
rHaPrP-res.sup.(Sc) band was detected in reactions seeded with a
6.times.10.sup.-8 dilution of ScBH containing .gtoreq.10 fg
(10.sup.-15 g) of PrP.sup.Sc (FIG. 2A). With a second round of
amplification seeded with 10% of the first round reaction products,
the sensitivity improved, allowing consistent detection of
dilutions of ScBH containing .about.50 ag (5.times.10.sup.-'.sup.7
g), or .about.1,000 molecules, of the original HaPrP.sup.Sc seed
(FIG. 2B). This amount of ScBH typically would contain an average
of 0.003 i.c. LD.sub.50 (a dose lethal to 50% of inoculated
hamsters) of scrapie infectivity according to 3 independent
end-point dilution bioassays of other brain homogenates stocks
prepared from Syrian hamsters in the clinical phase of scrapie
(Silveira et al. (2005) Nature 437, 257-261). A subset of replicate
reactions were positive with further dilutions of ScBH containing
.about.10-20 ag (nominally) of HaPrP.sup.Sc. However, none of the
NBH controls or samples seeded with more dilute ScBH gave
detectable 17-kDa bands. Further rounds of rPrP-PMCA did not
increase the sensitivity of PrP.sup.Sc detection. These results
indicate that rPrP-PMCA can detect sublethal amounts of
scrapie-infected tissue.
Example 5: Electron Microscopy and Fourier Transform Infrared
Spectroscopy (FTIR)
[0252] This Example describes electron microscopy and Fourier
transform infrared spectroscopy of rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon).
[0253] Negative-stained transmission electron microscopy of
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon) revealed that both
contained short bundles of fibrillar aggregates, which were
especially apparent after PK treatments (FIG. 8). However, other
than a tendency of rHaPrP-res.sup.(Sc) to be bundled laterally more
than rHaPrP-res.sup.(spon), we observed no consistent
ultrastructural differences between the two types of fibrils.
[0254] Comparisons of the secondary structures of
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon) by FTIR provided
additional evidence that they differ in conformation (FIG. 9). The
value of rHaPrP-res.sup.(Sc) as a PrP.sup.Sc surrogate will depend
in part upon the extent to which it mimics PrP.sup.Sc
conformationally. Comparison of rHaPrP-res.sup.(Sc) versus
rHaPrP-res.sup.(spon) showed that the former has a distinct
PK-resistant fragmentation pattern and an FTIR band at 1637
cm.sup.-1 (FIG. 9) that is reminiscent of 263K HaPrP.sup.Sc
itself.sup.23,24. There are also differences between the rPrP-res
fragment pattern and FTIR spectra of rHaPrP-res.sup.(Sc) and
HaPrP.sup.Sc. These differences could either be due to fundamental
conformational differences or to the lack of GPI anchor, N-linked
glycans, brain-derived ligands, or impurities in the rPrP-res.
Furthermore, it is not known whether rHaPrP-res.sup.(Sc) is
infectious, so caution should be used in interpreting
conformational analyses of rHaPrP-res.sup.(Sc). Nonetheless, the
data indicate that rHaPrP-res.sup.(Sc) is more closely related to
bona fide HaPrP.sup.Sc than is rHaPrP-res.sup.(spon).
Example 6: Competition Between rHaPrP-Res.sup.(Sc) and
rHaPrP-Res.sup.(spon)
[0255] This Example describes the competition between
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon) seen when reactions
are seeded with both rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon).
[0256] The effects of dual seeding of rPrP-PMCA reactions with both
rHaPrP-res.sup.(Sc) and rHaPrP-res.sup.(spon) were tested using
different seed ratios (FIG. 3). When the amounts of each seed were
equivalent, a mixture of the expected rHaPrP-res.sup.(Sc) and
rHaPrP-res.sup.(spon) reaction products was observed. However, when
one seed concentration was kept constant, addition of the other
seed reduced the formation of products expected from the first type
of seed. Excesses of 10- to 100-fold of one seed type nearly
eliminated the seeding activity of the other. This competition
and/or interference between the two types of seed makes it unlikely
that, once either rHaPrP-res.sup.(Sc) or rHaPrP-res.sup.(spon)
fibrils are prevalent in a reaction, the other could overtake the
reaction. This effect is probably due to competition for the
rPrP-sen substrate between mutually exclusive types of fibrils.
Example 7: Seeding with Cerebral Spinal Fluid (CSF)
[0257] This example demonstrates that CSF samples can be used to
discriminate uninfected and scrapie-affected hamsters by
rPrP-PMCA.
[0258] Because CSF is more accessible than brain tissue, rPrP-PMCA
seeding activity was compared in CSF samples collected from six
hamsters showing clinical signs of scrapie and three uninfected
control animals (all male). After one 24-hour round, no rHaPrP-res
was observed in the control reactions. However, all of the scrapie
CSF reactions produced the typical rHaPrP-res.sup.(Sc) banding
pattern with variable intensities (FIG. 4A). After second reactions
seeded with 10% of the volume of the first round reactions, the
control reactions each showed typical rHaPrP-res.sup.(spon)
patterns, while the scrapie-seeded reactions produced strong
rHaPrP-res.sup.(Sc) patterns of relatively uniform intensity (FIG.
4B). Analysis of CSF samples from 11 additional uninfected control
hamsters (2 females and 9 males) in a 2-round rPrP-PMCA gave either
no rHaPrP-res or the rHaPrP-res.sup.(spon) pattern. Thus, CSF
samples can be used to discriminate uninfected and scrapie-affected
hamsters by rPrP-PMCA.
Example 8: QUIC
[0259] This Example demonstrates that rPrP-PMCA can be carried out
in the form of an alternative assay referred to herein as QUIC
(quaking-induced conversion). In a QUIC assay, aggregates are
disrupted with periodic shaking of the reaction mix, rather than
(or in addition to) sonication.
[0260] Some laboratories have found the classical PMCA reaction to
be challenging to duplicate consistently, apparently due primarily
to difficulties in preparing the required brain homogenate
substrate preparations and delivering consistent sonication energy
to multiple reactions. To circumvent the aforementioned problems
with sonication, the QUIC assay was developed as a simplified and
more easily replicable method for sensitive PrP.sup.Sc and/or prion
detection. Like rPrP-PMCA, QUIC uses rPrP-sen as a substrate, but
substitutes periodic shaking for sonications. Even with this
modification, QUIC still can be approximately 10 times faster than
the current PMCA method that used brain homogenate as a source of
PrP-sen. The QUIC method is able to detect about 1 lethal
intracerebral scrapie dose within about 8 hours, and subinfections
doses with longer protocols. Under cell-free conditions with
intermittent shaking, sub-fentogram amounts of PrPSc in brain
homogenate and 2 .mu.l cerebral spinal fluid (CSF) from
scrapie-affected hamsters seeded the conversion of recombinant
prion protein into easily detectable quantities of specific
protease-resistant isoforms.
[0261] A solution of 0.2 mg/ml full-length bacterially expressed
hamster rPrP-sen (residues 23-231) was seeded with 10 ng of
purified hamster PrP.sup.Sc (263K strain) and the reaction
incubated for 25 hours with or without periodic shaking (FIG. 10A).
Treatment of the reaction products with proteinase K (PK) and
immunoblotting using an antiserum (R20) raised against a C-terminal
PrP epitope revealed PrP.sup.Sc-seeded PK-resistant conversion
products (rPrP-res.sup.(Sc)). Consistent with previous observations
with sonicated (rPrP-PMCA) reactions (described herein), QUIC
reactions produced prominent rPrP-res.sup.(Sc) bands of 17, 13, 12
and 10 kDa. Without shaking, the same rPrP-res.sup.(Sc) bands were
produced, but were much less intense.
[0262] Dilutions of scrapie brain homogenate were then seeded in
normal brain homogenate and the rPrPsen concentration and reaction
volume were varied (FIG. 10B). In 20-hour reactions, 100 .mu.l
reactions with 0.2 mg/ml rPrP-sen produced the most intense
rPrP-res.sup.(Sc) bands using seed dilutions containing as little
as 10 fg PrP.sup.Sc. Reactions seeded with only normal brain
homogenate produced either no PK-resistant products or a
spontaneously arising product(s), rPrP-res.sup.(spon), that gives a
set of 10-13 kD PK-resistant bands. The latter were similar to
those observed previously in unseeded rPrP-PMCA assays as described
herein. With 48-hour incubations at 0.2 mg/ml rPrP-sen, still
smaller amounts of scrapie brain homogenate seeded detectable
rPrP-res.sup.(Sc) in both 50- and 100-.mu.l reactions, with the
latter being more sensitive (FIG. 11A). Similar to previous
findings with rPrP-PMCA reactions, when the blot was probed with an
antibody to an epitope within PrP residues 96-106 (D13), the 17-kDa
band was stained preferentially. This indicated that the smaller
10-13 kDa bands that stained with the C-terminal antibody R20 were
C-terminal fragments that lacked the D13 epitope. With 65- and
95-hour incubations of 100 .mu.l reactions, seed dilutions
containing as little as 100 ag PrP.sup.Sc produced strong
rPrP-res.sup.(Sc) signals (FIG. 11).
[0263] In order to further improve sensitivity, two serial rounds
of QUIC reactions were performed in which products of a first
48-hour round were diluted into fresh rPrP-sen for a second-round
reaction (FIG. 12). In the first round, seeds nominally containing
as little as 25-50 ag of PrP.sup.Sc were frequently positive. After
second reactions seeded with 10% of the volume of the first round
reactions, more consistent detection of sub-femptogram amounts of
PrP.sup.Sc was observed with one of the 10-ag seeded samples being
positive for rPrP-res.sup.(Sc).
[0264] Because cerebral spinal fluid (CSF) is a more accessible
biopsy specimen than brain, rPrP-PMCA seeding activity was compared
in CSF samples collected from both hamsters showing clinical signs
of scrapie and uninfected control animals. After one 48-hour round,
no rHaPrP-res was observed in the control reactions. However, all
of the scrapie CSF reactions produced the typical
rHaPrP-res.sup.(Sc) banding pattern with variable intensities (FIG.
13). After the second serial reaction rounds, the control reactions
still lacked rPrP-res, while the reactions seeded with scrapie CSF
produced strong rHaPrP-res.sup.(Sc) patterns of relatively uniform
intensity. Thus, QUIC reactions seeded with CSF samples can
discriminate between uninfected and scrapie-affected hamsters.
[0265] Thus, QUIC provides a simple and easily duplicated
alternative to sonication for supporting an ultra-sensitive assay
for prions. With sonication of reaction tubes in cuphorn probes,
the delivery of vibrational energy to samples can vary
substantially and unpredictably with tube position, tube
construction, probe age, bath volume, and the redistribution of
samples within the tubes by sonication-induced atomization and
condensation. In contrast, when a group of sample tubes are shaken
in a rack, each tube is subjected to the same motion, making it
easier to treat all reactions equivalently. The sonicated rPrP-PMCA
reactions is somewhat faster and more sensitive than the shaken
QUIC reactions when both are performed at 37.degree. C., but
elevating the temperature of the QUIC reactions improves the speed
of the reaction and can shorten the overall assay length.
[0266] The observation that the QUIC assay can discriminate between
CSF samples taken from control and scrapie-affected hamsters
indicates that a diagnostic test for prion infections based on CSF
samples, as opposed to brain tissue, is feasible.
[0267] Testing of QUIC reaction conditions revealed that periodic
shaking enhanced PrP.sup.Sc seeded conversion of hamster rPrP-sen
(residues 23-231) into PK-resistant conversion products
[rPrP-res(Sc), where (Sc) refers to seeding by PrP.sup.Sc] (FIG.
10) which, consistent with our previous observations with sonicated
(rPrP-PMCA) reactions 7, produced prominent rPrP-res(Sc) bands of
17, 13, 12 and 11 kDa. Periodic shaking can therefore substitute
for sonication in promoting rPrP-res(Sc) formation. The
rPrP-res(Sc) generation was further improved by varying rPrP-sen
concentration, reaction volume (FIG. 10), reaction time (FIG. 11),
number of serial reactions (FIG. 12), temperature (FIG. 18), and
shaking cycle (FIG. 19). Furthermore, addition of the detergent
N-lauroyl sarcosine to the PK-digestion buffer improved the ratio
of the 17-kDa rPrP-res(Sc) band to the smaller bands (FIG. 20). In
QUIC reactions performed at 45.degree. C., rPrP-res(Sc) formed in
triplicate 1-round 46-h QUIC reactions seeded with .gtoreq.100-ag
of PrPSc (FIG. 14). In contrast, 21 negative control reactions
seeded with comparable dilutions of normal brain homogenate or
buffer alone produced no rPrP-res (FIG. 14). Results similar to
those in FIG. 1 were obtained in an identical repeat experiment
done in triplicate. When products of PrPSc-seeded reactions were
diluted 1000-fold into fresh rPrP-sen to seed the subsequent
reaction rounds, strong propagation of rPrP-res.sup.(Sc) through at
least 4 serial reactions was observed. Under some conditions, such
as with multiple serial 48-h reaction rounds at 45.degree. C.,
reactions seeded with only normal brain homogenate occasionally
generated a spontaneous product, rPrP-res.sup.(spon), indicated by
a set of .ltoreq.13 kDa PK-resistant bands. The latter were similar
to those observed previously in unseeded rPrP-PMCA assays and were
clearly distinct from the overall rPrP-res(Sc) banding profile.
Hence longer amplification assays, although they can detect very
small amounts of target in the sample, form more of the unwanted
rPrP-res.sup.(spon) product that competes with the desired
amplification reaction and that product could be confused with
rPrP-res.sup.(Sc) under some conditions.
[0268] Consistent with previous findings with rPrP PMCA reactions,
we found that when blots of PrPSc-seeded reaction products were
probed with an antibody to PrP residues 95-103 (D13)8, the 17-kDa
rPrP-res.sup.(Sc) band was stained preferentially (FIG. 10). This
result indicated that the smaller 11-13 kDa bands that reacted with
the C-terminal antibody R20 were C-terminal fragments lacking the
N-terminal portion of PrP containing the D13 epitope. Elevation of
QUIC reaction temperatures accelerated rPrP-res.sup.(Sc) formation
(FIG. 18). At 55.degree. C.), rPrP-res.sup.(Sc) was detected in
8-hour reactions seeded with as little as 10 fg PrPSc (.about.one
intracerebral infectious dose) (FIG. 18), while 1 fg could be
detected in triplicate 18-hours reactions (FIG. 21). At 65.degree.
C., 100 fg PrP.sup.Sc seed could be detected in only 4-hours (FIG.
18). However, at 65.degree. C., there was also more rapid formation
of rPrP-res.sup.(spon) in reactions seeded with normal brain
homogenate, which was apparent in all three reactions at 18 hours.
Overall, there is a tradeoff between sensitivity and speed in QUIC
assays and at any given temperature, the longer the total reaction
times the greater the likelihood of spontaneous (unseeded) rPrP-res
formation. However, spontaneous rPrP-res has usually produced
patterns of PK-resistant bands that are distinct from
rPrP-res.sup.(Sc). Interestingly, the patterns can be altered when
reaction conditions were pushed to both higher temperatures and
relatively long reaction times. The QUIC reaction conditions can be
altered to reduce the production of spontaneous rPrP-res that
appears similar to rPrP-res.sup.(Sc) according to the rPrP-sen
sequence used in the QUIC reaction.
[0269] Cerebral spinal fluid (CSF) is a more accessible biopsy
specimen than brain, hence QUIC seeding activity was evaluated in
CSF samples collected from hamsters showing clinical signs of
scrapie or uninfected control animals. After one 48-h round (at
37.degree. C.), no rHaPrP-res was seen in the control reactions.
However, all of the scrapie CSF reactions produced the distinctive
rHaPrP res.sup.(Sc) banding pattern albeit with variable
intensities (FIG. 17). After a second serial QUIC reaction, the
control reactions still lacked rPrP-res, while the reactions seeded
with scrapie CSF produced strong rHaPrP-res.sup.(Sc) patterns of
similar intensity. Similar 2-round QUIC reactions showed that CSF
samples from 10 additional uninfected control hamsters produced no
rHaPrP-res bands while two of the original scrapie-positive CSF
samples again produced strong rHaPrP-res.sup.(Sc) patterns (data
not shown). Thus, QUIC reactions seeded with CSF samples can
discriminate between uninfected and scrapie-affected hamsters.
[0270] A QUIC assay provides a simple alternative to sonication for
supporting an ultrasensitive prion assay. The delivery of
vibrational energy to samples does not vary substantially with tube
position, tube construction, probe age, bath volume, and the
redistribution of samples as often occurs within the tubes with
sonication-induced atomization and condensation. The 45.degree. C.
single-round QUIC reaction is virtually as sensitive as 2-round
sonicated rPrP-PMCA reactions of similar overall duration. The QUIC
reaction conditions are also less permissive of spontaneous
unseeded rPrP-res.sup.(spon) formation. Significantly, elevated
reaction temperatures can greatly accelerate QUIC reactions,
allowing detection of a lethal dose of 263K scrapie (i.c.) in <1
day (See FIGS. 15 and 18). The relative speed, simplicity and ease
of duplication of the QUIC reaction conditions offers major
practical advantages.
[0271] It is also possible to vary the shaking cycle to obtain
surprisingly superior results in the QUIC assay. For example, the
ratio of time spent shaking to time at rest can be varied to
improve the outcome of the assay. In some examples, the ratio of
time shaking:time at rest can vary from 1:15 to 1:1, such as 1:11
to 1:1. In particular examples, it has been found that
substantially equal periods of shaking and rest provide
particularly good results. For example, a shaking cycle of 60
seconds on and 60 seconds off works better than the 10 seconds on,
110 seconds off cycle for the hamster scrapie QUIC assay using
rPrP-sen 23-231.
[0272] The total length of a cycle (time spent shaking plus time
spent not shaking) may be less than about an hour, or even less
than 5 minutes, for example less then 3 minutes, such as 2 minutes
(120 seconds) or less. In particular examples, the total cycle is
more than 60 seconds, such as 60-180 seconds, or 60-120 seconds.
The shaking cycle can be optimized with regard to the rPrP-sen
sequence used in the QUIC reaction.
Example 9: Exemplary Protocol for rPrP-PCMA
[0273] This Example provides an exemplary step-by-step protocol for
rPrP-PMCA using hamster 263K scrapie seed and hamster rPrP-sen
substrate. Although specific exemplary protocols are provided, one
will appreciate that other similar protocols can be used.
I. Sample and Substrate Preparation
[0274] A. Preparation of Normal or 263K Scrapie Brain Homogenates
(NBH and ScBH, Respectively):
[0275] Reaction tubes were 0.2 ml thin wall PCR tube strips (Nalge
Nunc International 248161). Sample and substrate preparation was
carried out as follows: [0276] 1) Perfuse normal or
scrapie-affected Syrian golden hamsters with ice cold
1.times.PBS-EDTA:
TABLE-US-00009 [0276] NaCl 8 g KCl 0.2 g Na.sub.2PO.sub.4 1.44 g
KH.sub.2PO.sub.4 0.24 g +5 mM EDTA pH to 7.4 with HCl QS to 1 L
[0277] 2) Extract hamster brain with clean tools and flash freeze
with liquid nitrogen [0278] 3) Store perfused brains at -80.degree.
C. [0279] 4) Dounce homogenize perfused brains, on ice, in
conversion buffer (10% weight to volume):
TABLE-US-00010 [0279] 1X PBS-EDTA from step #1 (but 1 mM EDTA) 19.3
ml 5M NaCl 0.6 ml TRITON .RTM. X-100 0.1 ml Complete Protease
Inhibitor Cocktail, EDTA free 1 tablet/20 mls (Roche
11836170001)
[0280] 5) Spin NBH at 2000 g for 2 minutes to partially clarify;
collect supernatant [0281] 6) Prepare 1 ml 10% NBH aliquots and
flash freeze in liquid nitrogen [0282] 7) Store aliquots at
-80.degree. C.
[0283] B. Preparation of Hamster rPrP-sen:
Materials:
[0284] Approximately a 2 g cell pellet of rHaPrP 23-231 (yield from
1/4.sup.th of 1 L LB-Miller growth medium)
[0285] BugBuster.TM. and lysonase bioprocessing reagent (EMD
Biosciences)
[0286] 8M Guanidine in water
[0287] Ni-NTA Superflow resin (Qiagen)
[0288] Denaturing Buffer: 100 mM sodium phosphate, 10 mM Tris, 6M
Guanidine, pH 8.0
[0289] Refolding Buffer: 100 mM sodium phosphate, 10 mM Tris, pH
8.0
[0290] Elution Buffer: 500 mM imidazole, 10 mM Tris, 100 mM
Phosphate pH 5.8-6.0
[0291] Dialysis Buffer: 10 mM sodium phosphate, pH 6.5 (diluted
from 1M stock at pH 5.8)
[0292] AKTA Explorer 10 liquid chromatography system
Bacterial Cell Lysis:
[0293] 200 .mu.L of lysonase bioprocessing reagent and 1 complete
protease inhibitor tablet (Roche) were mixed into 50 mL of
BugBuster.TM. and stirred on ice. The frozen cell pellet was sliced
with a razor blade and added portionwise into the BugBuster.TM.
solution. Stirring was performed at 0.degree. C. while breaking up
larger pieces with a spatula. Sonication was performed for 15
second intervals with a Misonix ultrasonic cell disrupter (power
level 10) periodically over the course of .about.30 minutes until
the mixture was relatively homogeneous and became milky.
Centrifugation was carried out at at 10,000 g (JA 12 rotor, Beckman
Centrifuge) for 10 minutes. Pellets were washed twice with 20 mL
BugBuster.TM. diluted 10-fold with water, dispersed with
pipette-aid, and centrifuged at 10,000 g for 10 minutes. The
washing, dispersing and centrifuging was repeated, and the
inclusion body pellet was stored at -20.degree. C.
[0294] Purification was carried out by filling a 2.times.2 L
graduated cylinder with 10 mM phosphate dialysis buffer diluted
from 1 M stock at pH 5.8. All chromatography buffers were filtered
prior to use. The inclusion body pellet was dissolved into 8 mL of
8 M guanidine and mixed by pipetting up and down with a transfer
pipette. The mixture was transferred into 2 mL flip cap tubes and
centrifuged at 8,000 g for 10 minutes. Filter and wash fresh Ni-NTA
Superflow resin (Qiagen) exhaustively with water. Store dry at
4.degree. C. Then 18 g of Ni-NTA resin was weighed into a clean
beaker and the resin pre-equilibrated with 30-40 mL of denaturing
buffer by stirring at room temperature. Supernatant was added from
2 mL flip cap tubes to the resin and the tube discarded with the
pellet. Stirring was carried out for an additional 30 minutes. The
resin slurry was poured into an empty XK16/20 column and a column
attached with impregnated resin to an AKTA Explorer 10
(GE/Amersham) according to the manufacturer's directions. The
column outlet was detached and the flow-though collected directly
in a graduated cylinder. The flow-through can either be discarded
or saved for future use, as there is typically excess PrP in this
solution.
[0295] A linear gradient was run with 0-100% refold buffer at 0.75
mL/min over 5-6 hours, followed by 100% refolding buffer for 30-60
minutes at 1 mL/min. The pump were rinsed with distilled water, and
then Line A equipped with refold buffer and Line B with elution
buffer. The bottom of column was reattached to the UV and
conductivity detector. Elution buffer was run through line B and
the UV autozero detector set at 280 nm. The refolded peptide was
eluted at 2 mL/min for 20 minutes. After a brief forerun, the major
fraction was collected at UV 280 as one portion in a 250 mL
graduated cylinder prefilled with 50 mL pure water. The protein was
diluted with water to 150 mL, then sterile filtered with a 150 mL
filter unit. An expected concentration of .about.0.1-0.15 mg/mL is
determined by A.sub.280. The protein was dialyzed (Snakeskin
dialysis tubing MWCO 7000) overnight in dialysis buffer, and the
protein transferred into fresh dialysis buffer for 1 hour. If there
was any turbidity at this point, immediate sterile filtration was
performed. The peptide was analyzed for purity by SDS-PAGE, Western
blot, and MALDI, and the protein concentrated to .about.0.4 mg/ml
in 10 mM sodium phosphate buffer, pH 6.5, using an Amicon Ultracel
-10 k filter (15 ml capacity). Aliquots were flash frozen and
stored at -80.degree. C. Once thawed, it was kept at 4.degree.
C.
[0296] C. 4.times.PMCA Buffer
[0297] (Final composition: 0.2% SDS, 0.2% TRITON.RTM. X-100,
4.times.PBS)
[0298] 10% SDS stock (20 .mu.l/ml)
[0299] 10% TRITON.RTM. X-100 stock (20 .mu.l/ml)
[0300] 10.times.PBS stock (400 .mu.l/ml):
TABLE-US-00011 Na.sub.2HPO.sub.47H.sub.20 26.8 g/L
NaH.sub.2PO.sub.4H.sub.20 13.8 g/L NaCl 75.9 g/L pH 6.9 H.sub.2O
(560 .mu.l/ml)
II. rPrP-PMCA Protocol:
[0301] A. 1st rPrP-PMCA Round was Carried Out According to this
Protocol:
[0302] 1) Sonicator setup: Misonix 3000 with microplate (cup)horn
accessory
[0303] 2) Circulating water bath was set up at 39.4 degrees for cup
horn, resulting in a temperature of 37 degrees in the cup horn.
[0304] 3) 1 ml of 1.times.PMCA buffer was made up from 4.times.
stock
[0305] 4) Thawed aliquots of 10% NBH & 10% ScBH
[0306] 5) Centrifuged 10% NBH at 1000 rcf for 5 minutes at
4.degree. C. to remove large debris.
[0307] 6) Made up 1 ml of 1% NBH by dilution into 1.times.PMCA
buffer
[0308] 7) Prepared 263K BH seed diluted in 1% NBH (see dilution
series below) [0309] a) dilute 10% 263K BH 1:20 into 1% NBH (5
.mu.l stock+9 .mu.l 1% NBH).fwdarw.500 pg/1 .mu.l [0310] b) further
dilute 1:10 (5 .mu.l previous+45 .mu.l 1% NBH).fwdarw.50 pg/1 .mu.l
[0311] c) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 pg/1 .mu.l [0312] d) further dilute 1:10 (5 .mu.l
previous+45 .mu.l 1% NBH).fwdarw.500 fg/1 .mu.l [0313] e) further
dilute 1:10 (5 .mu.l previous+45 .mu.l 1% NBH).fwdarw.50 fg/1 .mu.l
[0314] f) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 fg/1 .mu.l [0315] g) further dilute 1:10 (5 .mu.l
previous+45 .mu.l 1% NBH).fwdarw.500 ag/1 .mu.l [0316] h) further
dilute 1:10 (5 .mu.l previous+45 .mu.l 1% NBH).fwdarw.50 ag/1 .mu.l
[0317] i) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 ag/1 .mu.l [0318] j) further dilute 1:10 (5 .mu.l
previous+45 .mu.l 1% NBH).fwdarw.0.5 ag/1 .mu.l [0319] (1 fg=2
.mu.l of g) [0320] (100 ag=2 .mu.l of h) [0321] (30 ag=6 .mu.l of
i) [0322] (10 ag=2 .mu.l of i)
[0323] 8) Prepared reaction mix in reaction tubes as described
above (adding in the order specified)
[0324] 1.sup.st Round Reaction Mix:
TABLE-US-00012 31.3 .mu.l H2O 20 .mu.l 4X PMCA buffer 26.7 .mu.l
rPrP-sen (to give a final concentration of 0.1 mg/ml) 2 .mu.l ScBH
seed diluted in 1% NBH 80 .mu.l total volume
[0325] 9) When adding the rPrP-sen and then the seed material to
the 1.times.PMCA buffer in the reaction mix, mixing was performed
by pipetting up and down gently without vortexing. The reaction
tubes were capped but without vortexing. Place tube strips were
placed in a floating 96 well rack in the sonicator cup horn, cover
cup with plastic wrap to reduce splashes and evaporation.
[0326] 10) Started sonicator program (typical: 40 second
intermittent sonication at power setting #10, 16 minute total
sonication time, 59 minute 20 second incubations between each
sonication, 24 hour total cycle time)
[0327] 11) Following sonication cycle (24 hours), turned off
sonicator and removed tube strips.
[0328] 12) Spun the tube strips briefly to bring solution down out
of the caps.
[0329] 13) Removed aliquot for 2.sup.nd rPrP-PMCA round and/or
prepared for methanol precipitation and immunoblot analysis (see
below).
[0330] B. 2nd PMCA Round:
[0331] 1) Prepare reaction mix in fresh reaction tube strips as
described for 1st round above. The sample was gently vortexed to
evenly suspend just prior to transferring volume. Following the
addition of rPrP-sen, mixing was performed by pipetting up and
down.
2nd Round Reaction Mix:
TABLE-US-00013 [0332] 30 .mu.l H.sub.2O 18 .mu.l 4X PMCA buffer 24
.mu.l rPrP-sen (a volume to give a final concentration of 0.1
mg/ml) 8 .mu.l reaction aliquot from first round rPrP-PMCA
[0333] 2) The reaction tube strips were capped, and the tube strips
placed in the floating rack in the sonicator cup horn. The cup horn
was covered with plastic wrap to reduce splashes and evaporation.
The sonicator program was started (typical: 40 second intermittent
sonication at #10, 16 minute total sonication time, 59 minute 20
second incubations between each sonication, 24 hours total cycle
time). Following the sonication cycle (24 hours), the sonicator was
turned off and tube strips removed. The tube strips were quick spun
to bring solution down out of the caps, and the samples were
methanol precipitated prior to further analysis (see below).
[0334] C. PK-Digestion and SDS-PAGE Sample Preparation:
[0335] (Note: In the following example, the methanol
precipitation-associated steps 7-11 can often be omitted, in which
case the products of step 6 are mixed directly with more
concentrated SDS-PAGE sample buffer)
[0336] 1) Prepared 0.1% SDS in 1.times.PBS
[0337] 2) Transferred 5 .mu.l of each sample to a clean screw cap
tube. (vortexed sample to evenly suspend just prior to transferring
volume)
[0338] 3) Added 19 .mu.l 0.1% SDS in PBS
[0339] 4) Added 1 .mu.l 75 .mu.g proteinase K (PK)/ml (final
concentration will be 3 .mu.g PK/ml) PK storage buffer [0340] PK
storage buffer: [0341] 50% glycerol [0342] 1 mM CaCl2 [0343] 50 mM
Tris, pH 8.5
[0344] 5) Incubated at 37 degrees for 1 hour
[0345] 6) Added 1 .mu.l of 0.1M PEFABLOC.RTM.
(4-(2-Aminoethyl)-benzensulfonyl fluoride) (Roche), vortex and
place on ice
[0346] 7) Added 4 .mu.l of thyroglobulin (5 mg/ml), vortex and keep
on ice
[0347] 8) Added 120 .mu.l (4 volumes) of cold methanol, vortexed
and kept on ice
[0348] 9) Stored at -20 degrees for .gtoreq.1 hour
[0349] 10) Spun at 20800 rcf in the EPPENDORF.RTM. 5417R centrifuge
at 4 degrees for 30 minutes
[0350] 11) Aspirated off supes and leave caps off to air dry
samples
[0351] 12) Added 15 .mu.l 1.times.SDS-PAGE sample buffer containing
4M Urea to each tube
[0352] 13) Vortexed samples in SDS-PAGE sample buffer for 1
minute
[0353] 14) Boiled tubes for 10 minutes
[0354] 15) Loaded sample onto 10% NUPAGE.RTM. (polyacrylamide) gel
& run
[0355] D. Immunoblotting:
[0356] Wet transfer was performed using Towbin transfer buffer,
IMMOBILON.RTM.-P Blotting sandwiches (transfer membrane, Millipore
IPSN07852) and BIORAD MINI TRANS-BLOT.RTM. for 1 hour at 0.3 amps
constant. The primary antibodies used were R20 (J. Virol. 65,
6597-6603 (1991)) at 1:30,000 or D13 (Nature 412, 739-743 (2001))
at 1:10,000. The secondary antibodies were anti-rabbit or
anti-human AP conjugated, as appropriate Immunostaining was
visualized by ATTOPHOS.RTM. AP Fluorescent Substrate System
(Promega) (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate
[BBTP]) according to the manufacturer's recommendations.
III. QUIC Protocol
[0357] A. 1.sup.st QUIC Round:
[0358] 1 ml of 1.times.PMCA buffer was made up from 4.times. stock,
and aliquots of 10% NBH & 10% ScBH were thawed. Centrifuged 10%
NBH at 2000 rcf for 2 minutes at 4.degree. C. to remove large
debris. Made up 1 ml of 1% NBH by dilution into 1.times.PMCA buffer
and prepared 263K BH seed diluted in 1% NBH (see dilution series
below).
263K BH Seed Dilution Series:
[0359] a) dilute 10% 263K BH 1:20 into 1% NBH (5 .mu.l stock+95
.mu.l 1% NBH).fwdarw.500 pg/1 .mu.l
[0360] b) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.50 pg/1 .mu.l
[0361] c) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 pg/1 .mu.l
[0362] d) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.500 fg/1 .mu.l
[0363] e) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.50 fg/1 .mu.l
[0364] f) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 fg/1 .mu.l
[0365] g) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.500 ag/1 .mu.l
[0366] h) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.50 ag/1 .mu.l
[0367] i) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.5 ag/1 .mu.l
[0368] j) further dilute 1:10 (5 .mu.l previous+45 .mu.l 1%
NBH).fwdarw.0.5 ag/1 .mu.l [0369] (1 fg=2 .mu.l of g) [0370] (100
ag=2 .mu.l of h) [0371] (30 ag=6 .mu.l of i) [0372] (10 ag=2 .mu.l
of i)
[0373] Prepared reaction mix in reaction tubes as described above
(add in the order specified).
1.sup.st Round Reaction Mix:
[0374] 47.4 .mu.l H2O [0375] 25 .mu.l 4.times.PMCA buffer [0376]
25.6 .mu.l rPrP-sen [0377] 2 .mu.l ScBH seed diluted in 1% NBH
[0378] 100 .mu.l total volume
[0379] Adjusted H.sub.2O and rPrP-sen volumes to give a final
rPrP-sen concentration of 0.1 mg/ml. When adding the rPrP-sen and
then the seed material to the 1.times.PMCA buffer in the reaction
mix, mixing was performed by pipetting up and down gently without
vortexing. The reaction tubes were capped but not vortexed. The
tubes were placed in EPPENDORF THERMOMIXER.RTM. R with 24.times.0.5
ml tube block and incubated in Thermomixer R for the desired time
at 37.degree. C., alternating between 10 seconds of shaking at 1500
rpm and no shaking for 110 seconds, unless designated otherwise.
The tubes were spun to bring any solution down out of the caps. An
aliquot was removed for 2nd QUIC round and/or prepared for PK
digestion, methanol precipitation and immunoblot analysis (see
below).
[0380] B. 2nd QUIC Round:
[0381] The reaction mix was prepared in fresh reaction tubes
similar to 1.sup.st round above. The sample tubes were gently
vortexed to evenly suspend any pellet just prior to transferring
volume to the 2.sup.nd round reaction tube. Following the addition
of rPrP-sen, mixed by pipetting up and down.
2.sup.nd Round Reaction Mix:
[0382] 43.5 .mu.l H2O [0383] 22.5 .mu.l 4.times.PMCA buffer [0384]
24 .mu.l rPrP-sen [0385] 10 .mu.l sample volume from 1.sup.st round
reaction [0386] 100 .mu.l total volume
[0387] The H.sub.2O and rPrP-sen volumes were adjusted to give a
final rPrP-sen concentration of 0.1 mg/ml. When adding the rPrP-sen
and then the seed material to the 1.times.PMCA buffer in the
reaction mix, mixing was performed by pipetting up and down gently
without vortexing. The seed was diluted in 1% NBH as described in
the 1.sup.st QUIC round, and the remainder of the method performed
as in 1.sup.st QUIC round.
PK-Digestion and SDS-PAGE Sample Preparation:
[0388] (Note: In the following, the methanol
precipitation-associated steps 7-11 can often be omitted, in which
case the products of step 6 is mixed directly with more
concentrated SDS-PAGE sample buffer)
[0389] 1) Prepared 0.1% SDS in 1.times.PBS
[0390] 2) Transferred 10 .mu.l of each sample to a clean screw cap
tube and vortexed sample to evenly suspend any pellet just prior to
transferring volume
[0391] 3) Added 38 .mu.l 0.1% SDS in PBS
[0392] 4) Added 2 .mu.l 75 .mu.g proteinase K (PK)/ml (final
concentration will be 3 .mu.g PK/ml) PK storage buffer (50%
glycerol, 1 mM CaCl.sub.2, 50 mM Tris, pH 8.5)
[0393] 5) Incubated at 37 degrees for 1 hour
[0394] 6) Added 1 .mu.l of 0.1M PEFABLOC.RTM.
(4-(2-Aminoethyl)-benzensulfonyl fluoride) (Roche), vortexed and
placed on ice
[0395] 7) Added 4 .mu.l of thyroglobulin (5 mg/ml), vortexed and
kept on ice
[0396] 8) Added 120 .mu.l (4 volumes) of cold methanol, vortexed
and kept on ice
[0397] 9) Stored at -20 degrees for .gtoreq.1 hour
[0398] 10) Spun at 20800 rcf in EPPENDORF.RTM. 5417R centrifuge at
4 degrees for 30 minutes
[0399] 11) Aspirated off supernatant and left caps off to air dry
samples
[0400] 12) Added 15 .mu.l 1.times.SDS-PAGE sample buffer containing
4M Urea to each tube
[0401] 13) Vortexed samples in SDS-PAGE sample buffer for 1
minute
[0402] 14) Boiled tubes for 10 minutes
[0403] 15) Loaded sample onto 10% NUPAGE.RTM. (polyacrylamide) gel
& run
Immunoblotting:
[0404] Wet transferred using Towbin transfer buffer,
IMMOBILON.RTM.-P Blotting sandwiches (transfer membrane, Millipore
IPSN07852) and BioRad MINI TRANS-BLOT.RTM. for 1 hour at 0.3 amps
constant.
[0405] Primary antibodies: R20 [J. Virol. 65, 6597-6603 (1991)] at
1:30,000 or D13 [Nature 412, 739-743 (2001)] at 1:10,000.
[0406] Secondary antibodies anti-rabbit or anti-human AP
conjugated, as appropriate.
[0407] Immunostaining was visualized by ATTOPHOS.RTM. AP
Fluorescent Substrate System (Promega)
(2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate [BBTP])
according to the manufacturer's recommendations.
Example 10: Amplification of PrP-res from a Variant-CJD (vCJD)
Patient
[0408] FIG. 20 shows Western blots from a QUIC reaction seeded
either with dilutions of a brain homogenate (BH) from human variant
CJD patient (vCJD BH) containing 100 fg, 10 fg, or 1 fg of PrP-res
or, as a negative control, a dilution of a non-CJD human brain
homogenate (from an Alzheimer's patient; AD-BH) equivalent to the
100-fg vCJD brain dilution. The recombinant PrP (rPrP-sen)
substrate in these reactions was the Syrian hamster PrP sequence
(residues 23-231). A single-round reaction was performed at
50.degree. C. for either 8 hours (top blots) or 18 hours (bottom
blots). The primary antiserum used to detect the rPrP-res[CJD]
reaction products was R20. Six separate reactions were performed
with each type or dilution of seed and the number of
rHaPrP-res.sup.(vCJD)-positive reactions per 6 replicates is
indicated below each set of replicates on the blots.
[0409] vCJD-BH dilutions containing a nominal 100 fg of PrP-res
produced clear rHaPrP-res.sup.(vCJD) patterns in five out of six
8-h reactions, and in 6/6 18-hour cross-species QUIC reactions.
Samples with 10 fg PrP-res were positive for rHaPrP-res.sup.(vCJD)
in 5/6 reactions of both 8 and 18 h. Samples with 1 fg PrP-res were
rHaPrP-res.sup.(vCJD)-positive in 1/6 8-h and 2/6 18-h reactions.
At the same time, the AD-BH gave no rHaPrP-res.sup.(vCJD)-positive
reactions with either reaction time. Although it is unknown how
much PrP-res is required for an infectious dose, it is known that a
lethal intracerebral dose of hamster 263K scrapie usually
corresponds to 1-10 fg of PrP.sup.Sc. Thus, this cross-species QUIC
reaction can detect quantities of vCJD PrP-res (as little at 10 fg
and even as low as 1 fg) that approximate that of an infectious
dose of scrapie by the most efficient intracerebral route.
[0410] The assay was carried out in 0.5 ml conical microcentrifuge
tubes with screw caps (Fisher 02-681-334). Brains were homogenized
in conversion buffer (10% weight to volume):
TABLE-US-00014 1X PBS-EDTA from step #1 (but 1 mM EDTA) 19.3 ml 5M
NaCl 0.6 ml TRITON .RTM. X-100 0.1 ml Complete Protease Inhibitor
Cocktail, EDTA free 1 tablet/20 mls (Roche 11836170001)
[0411] Brain homogenates (BH) were spun spin at 2000 g for 2
minutes to partially clarify; supernatant was collected and 1 ml
10% AD-BH and vCJD-BH aliquots were prepared and frozen for storage
at -80.degree. C.
[0412] Hamster rPrP-sen was prepared as in Example 8, as were
bacterial cell lysis and purification. The same 4.times.PMCA buffer
was used as in Example 8. The QUIC protocol was carried out by
making up a working stock of 0.1% SDS in 1.times.PBS, thawing
aliquots of 10% AD-BH & 10% vCJD-BH, making up 1 ml of
1.times.N2 supplement (Invitrogen) by dilution into 0.1% SDS/PBS.
The AD-BH & vCJD-BH seed dilutions in 1.times.N2 were carried
out as follows: [0413] 7.1 ul 10% AD-BH or 10% vCJD-BH+2.9 ul
1.times.N2.fwdarw.1 ug/2 ul [0414] further dilute 1:10 (5 ul
previous+45 ul 1.times.N2).fwdarw.100 pg/2 ul [0415] further dilute
1:10 (5 ul previous+45 ul 1.times.N2).fwdarw.10 pg/2 ul [0416]
further dilute 1:10 (5 ul previous+45 ul 1.times.N2).fwdarw.1 pg/2
ul [0417] further dilute 1:10 (5 ul previous+45 ul
1.times.N2).fwdarw.100 fg/2 ul [0418] further dilute 1:10 (5 ul
previous+45 ul 1.times.N2).fwdarw.10 fg/2 ul [0419] further dilute
1:10 (5 ul previous+45 ul 1.times.N2).fwdarw.1 fg/2 ul Recombinant
PrP was filtered with a 100 kD microtube filter (PALL) by spinning
at 3000.times.g for 12 min, and diluted 1:10 in 0.1% SDS/PBS and
measured spectrometrically for optical density at 280 nm.
[0419] [Protein] mg/mL=(280 nm reading/PrP Extinction Coefficient
(2.6))*Dilution Factor=X mg/mL [0420] Want 0.1 mg/mL rPrP in 100 uL
reaction=10 ug/X=Y uL rPrP per reaction [0421] Amount of water in
reaction=100-Y-2-25=Z uL Water per reaction The reaction mix was
prepared in reaction tubes as described above (add in the order
specified).
[0422] 1.sup.st Round Reaction Mix:
TABLE-US-00015 .sup. Z ul H.sub.2O 13 ul 25 ul 4X QUIC buffer 25 ul
2 ul ScBH seed diluted in 1% NBH 2 ul Y ul rPrP-sen 60 ul 100 ul
total volume
The first three components were vortexed for 5 s prior to adding
the rPrP-sen, and the rPrP-sen was added gently, as not to create
bubbles. The reaction tubes were capped but not vortexed. The tubes
were placed in an EPPENDORF THERMOMIXER.RTM. with 24.times.0.5 ml
tube block and incubated for the designated time (either 8 or 18
hrs) at 50.degree. C., alternating between 60 seconds of shaking at
1500 rpm and no shaking for 60 sec. The Thermomixer R is programmed
to adjust to 4.degree. C. following the 50.degree. C. time.
Spinning of the tubes was performed to recover any solution from
the caps.
[0423] PK-digestion and SDS-PAGE sample preparation were performed
by preparing 1% N-lauroylsarcosine sodium salt (sarkosyl) in
1.times.PBS, and diluting stock proteinase K (PK) (10 mg/ml)
100-fold into PK storage buffer (final concentration will be 100 ug
PK/ml). [0424] PK storage buffer: [0425] 50% glycerol [0426] 1 mM
CaCl.sub.2 [0427] 50 mM Tris, pH 8.5 Further diluted 100 .mu.g
PK/ml solution above 1 to 5 in 1% Sarkosyl/PBS (25 ul+100 ul 1%
Sarkosyl/PBS), transferred 5 .mu.l of PK/Sarkosyl solution to a
fresh set of tube, and vortexed QUIC sample tubes evenly to suspend
any pellet just prior to transferring volume, then transferred 10
ul to individual tubes containing PK/Sarkosyl. Incubation was
performed at 37.degree. C. for 1 hour, then 15 .mu.l
12.times.SDS-PAGE sample buffer containing 4M Urea was added to
each tube. The samples were vortexed in SDS-PAGE sample buffer for
1 minute, the tubes boiled for 10 minutes, and the samples
subjected to zip spinning and loaded onto 10% NUPAGE.RTM.
polyacrylamide gel (Invitrogen) with MES buffer (Invitrogen).
[0428] Immunoblotting was performed by pre-incubating membranes in
methanol for 3 minutes to wet the PVDF membrane, pouring off the
methanol and adding Towbin buffer to the VDF membrane. Dry transfer
was performed using Invitrogen iGel System and IMMOBILON-P.RTM.
polyvinylidene fluoride (PVDF) membrane (Millipore IPSN07852) for 7
minutes. The membrane was blocked in 5% Milk/TBST at room
temperature for 30 minutes. It was exposed to primary antibody (R20
at 1:10,000 (2 uL/20 mL 5% Milk/TBST) for 30 min at room
temperature) then washed 3.times. in .about.30 mL TBST (500 uL
Tween 20/1 L 1.times.TBS) for 5 minutes per wash. The secondary
antibody was Goat anti-rabbit-AP conjugate (1:10,000 in 5%
milk/TBST or 2 uL/20 mL) (Jackson) for 30 minutes). Washing was
performed 3.times. in TBST for 5 minutes per wash. Then 1.5 mL
ATTOPHOS.RTM. AP (alkaline phosphatase) Fluorescent Substrate
System (Promega) (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole
phosphate [BBTP]) was added to the plastic container and gel placed
face down onto it for 4 minutes, following which the gel was
removed and left on its edge to dry. The gel was visualized on a
STORM.TM. imaging system (Amersham).
Example 11
Amplification of PrP from Sheep and Cows
[0429] Sheep with nervous disorders resembling those of a scrapie
infection are purchased or donated. In some cases, sheep are
diagnosed with scrapie by histopathological and immunohistochemical
examination of the brain. If necropsy is performed, it is performed
within 36 hours after natural death or immediately after killing
the animal by intravenous injection of sodium pentobarbital and
exsanguination. The brain is removed from each sheep for scrapie
diagnosis. Blood, serum, cerebral spinal fluid and/or brain tissue
samples are obtained from each sheep.
[0430] Cows with nervous system disorders resembling those of
bovine spongiform encephalitis are also tested. These animals can
be "downers" or can exhibit less severe symptoms. In some cases,
animals that appear healthy can be tested to determine that they
are not infected.
[0431] The samples are used to seed the conversion of rPrP-sen to
protease-resistant forms in reactions performed in 0.1% sodium
dodecyl sulfate and 0.1% TRITON.RTM. X-100, in PBS at 37.degree. C.
in 0.5 ml tubes. Tube shaking is done at 1500 rpm in an EPPENDORF
THERMOMIXER.RTM. R or by vortexing. Proteinase K digestions and
immunoblotting were performed as described above.
[0432] For comparing PK-resistant QUIC reaction products, 24-hour
unshaken reactions and reactions were shaken with or without 0.1 mm
glass cell disruption beads (Scientific Industries). These
reactions are seeded with 0.2 mg/ml hamster rPrP-sen, 0.2 mg/ml
bovine rPrP-sen, or 0.2 mg/ml sheep rPrP-sen and a 50 .mu.l
reaction volume. The tubes are subjected to cycles of 2 minutes of
shaking and 28 minutes without shaking. C-terminal antibody R20 is
used for the immunoblot. The tubes are subjected to cycles of 10
seconds of shaking and 110 seconds without shaking. R20 was used
for the immunoblot.
[0433] If needed 65-hour and 95-hour QUIC reactions are carried out
as described above, and 0.2 mg/ml rPrP-sen, is used for 100-.mu.l
reaction volumes. Cycles of 10 seconds shaking and 110 seconds
without shaking can be used.
[0434] In other examples, 48-hour reaction times are used with
reduced detergent concentrations (0.05% SDS and 0.05% TRITON.RTM.
X-100). For the second round, 10% of the volume of the first round
reaction products are diluted into 9 volumes of reaction buffer
containing fresh rPrP-sen. PK-digestions and immunoblotting using
either R20 or D13 primary antibodies were performed as described
above.
[0435] For seeding with CSF samples, aliquots (2 .mu.l) of CSF are
used to seed QUIC reactions using the conditions, and immunoblots
are carried out using the PK-digested products of the first 48-hour
round. Ten percent of each first round reaction volume is used to
seed a second 48-hour round of QUIC. Antibodies R20 and D13 are
used for the immunoblots.
[0436] The foregoing examples provide specific examples of methods
for carrying out the disclosed assay. In view of the many possible
embodiments to which the principles of the disclosed assay can be
applied, it should be recognized that the illustrated embodiments
should not be taken as a limitation on the scope of the disclosure.
Rather, the scope of the disclosure is defined by the following
claims. We therefore claim all that comes within the scope and
spirit of these claims.
Sequence CWU 1
1
111209PRTMesocricetus auratus 1Lys Lys Arg Pro Lys Pro Gly Gly Trp
Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser Pro Gly
Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Thr Trp Gly Gln
Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly Gly Trp
Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 50 55 60 Gly
Gly Trp Gly Gln Gly Gly Gly Thr His Asn Gln Trp Asn Lys Pro 65 70
75 80 Asn Lys Pro Lys Thr Ser Met Lys His Met Ala Gly Ala Ala Ala
Ala 85 90 95 Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly
Ser Ala Met 100 105 110 Ser Arg Pro Met Leu His Phe Gly Asn Asp Trp
Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu Asn Met Asn Arg Tyr Pro Asn
Gln Val Tyr Tyr Arg Pro Val 130 135 140 Asp Gln Tyr Asn Asn Gln Asn
Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160 Thr Ile Lys Gln
His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170 175 Thr Glu
Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180 185 190
Val Thr Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg 195
200 205 Ser 2208PRTMus musculus 2Lys Lys Arg Pro Lys Pro Gly Gly
Trp Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser Pro
Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Thr Trp Gly Gln
Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly 35 40 45 Ser Trp
Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly 50 55 60
Gly Trp Gly Gln Gly Gly Gly Thr His Asn Gln Trp Asn Lys Pro Ser 65
70 75 80 Lys Pro Lys Thr Asn Leu Lys His Val Ala Gly Ala Ala Ala
Ala Gly 85 90 95 Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly
Ser Ala Met Ser 100 105 110 Arg Pro Met Ile His Phe Gly Asn Asp Trp
Glu Asp Arg Tyr Tyr Arg 115 120 125 Glu Asn Met Tyr Arg Tyr Pro Asn
Gln Val Tyr Tyr Arg Pro Val Asp 130 135 140 Gln Tyr Ser Asn Gln Asn
Asn Phe Val His Asp Cys Val Asn Ile Thr 145 150 155 160 Ile Lys Gln
His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr 165 170 175 Glu
Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys Val 180 185
190 Thr Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg Ser
195 200 205 3209PRTHomo sapiens 3Lys Lys Arg Pro Lys Pro Gly Gly
Trp Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser Pro
Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp Gly
Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly Gly
Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 50 55 60
Gly Gly Trp Gly Gln Gly Gly Gly Thr His Ser Gln Trp Asn Lys Pro 65
70 75 80 Ser Lys Pro Lys Thr Asn Met Lys His Met Ala Gly Ala Ala
Ala Ala 85 90 95 Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu
Gly Ser Ala Met 100 105 110 Ser Arg Pro Ile Ile His Phe Gly Ser Asp
Tyr Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu Asn Met His Arg Tyr Pro
Asn Gln Val Tyr Tyr Arg Pro Met 130 135 140 Asp Glu Tyr Ser Asn Gln
Asn Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160 Thr Ile Lys
Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170 175 Thr
Glu Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180 185
190 Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly Ser
195 200 205 Ser 4209PRTHomo sapiens 4Lys Lys Arg Pro Lys Pro Gly
Gly Trp Asn Thr Gly Gly Ser Arg Tyr 1 5 10 15 Pro Gly Gln Gly Ser
Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly 20 25 30 Gly Gly Trp
Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 35 40 45 Gly
Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 50 55
60 Gly Gly Trp Gly Gln Gly Gly Gly Thr His Ser Gln Trp Asn Lys Pro
65 70 75 80 Ser Lys Pro Lys Thr Asn Met Lys His Met Ala Gly Ala Ala
Ala Ala 85 90 95 Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Val Leu
Gly Ser Ala Met 100 105 110 Ser Arg Pro Ile Ile His Phe Gly Ser Asp
Tyr Glu Asp Arg Tyr Tyr 115 120 125 Arg Glu Asn Met His Arg Tyr Pro
Asn Gln Val Tyr Tyr Arg Pro Met 130 135 140 Asp Glu Tyr Ser Asn Gln
Asn Asn Phe Val His Asp Cys Val Asn Ile 145 150 155 160 Thr Ile Lys
Gln His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe 165 170 175 Thr
Glu Thr Asp Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys 180 185
190 Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly Ser
195 200 205 Ser 5218PRTBos taurus 5Lys Lys Arg Pro Lys Pro Gly Gly
Gly Trp Asn Thr Gly Gly Ser Arg 1 5 10 15 Tyr Pro Gly Gln Gly Ser
Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly 20 25 30 Gly Gly Gly Trp
Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 35 40 45 Gly Gly
Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 50 55 60
Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Gly Trp Gly Gln Gly 65
70 75 80 Gly Thr His Gly Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr
Asn Met 85 90 95 Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val
Val Gly Gly Leu 100 105 110 Gly Gly Tyr Met Leu Gly Ser Ala Met Ser
Arg Pro Leu Ile His Phe 115 120 125 Gly Ser Asp Tyr Glu Asp Arg Tyr
Tyr Arg Glu Asn Met His Arg Tyr 130 135 140 Pro Asn Gln Val Tyr Tyr
Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn 145 150 155 160 Asn Phe Val
His Asp Cys Val Asn Ile Thr Val Lys Glu His Thr Val 165 170 175 Thr
Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys Met 180 185
190 Met Glu Arg Val Val Glu Gln Met Cys Ile Thr Gln Tyr Gln Arg Glu
195 200 205 Ser Gln Ala Tyr Tyr Gln Arg Gly Ala Ser 210 215
6209PRTOvis aries 6Lys Lys Arg Pro Lys Pro Gly Gly Gly Trp Asn Thr
Gly Gly Ser Arg 1 5 10 15 Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn
Arg Tyr Pro Pro Gln Gly 20 25 30 Gly Gly Gly Trp Gly Gln Pro His
Gly Gly Gly Trp Gly Gln Pro His 35 40 45 Gly Gly Gly Trp Gly Gln
Pro His Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly Gly Gly
Trp Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys 65 70 75 80 Pro Ser
Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 85 90 95
Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala 100
105 110 Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu Asp Arg
Tyr 115 120 125 Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val Tyr
Tyr Arg Pro 130 135 140 Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val
His Asp Cys Val Asn 145 150 155 160 Ile Thr Val Lys Gln His Thr Val
Thr Thr Thr Thr Lys Gly Glu Asn 165 170 175 Phe Thr Glu Thr Asp Ile
Lys Ile Met Glu Arg Val Val Glu Gln Met 180 185 190 Cys Ile Thr Gln
Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly 195 200 205 Ala
7210PRTCervus unicolor 7Lys Lys Arg Pro Lys Pro Gly Gly Gly Trp Asn
Thr Gly Gly Ser Arg 1 5 10 15 Tyr Pro Gly Gln Gly Ser Pro Gly Gly
Asn Arg Tyr Pro Pro Gln Gly 20 25 30 Gly Gly Gly Trp Gly Gln Pro
His Gly Gly Gly Trp Gly Gln Pro His 35 40 45 Gly Gly Gly Trp Gly
Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 50 55 60 Gly Gly Gly
Gly Trp Gly Gln Gly Gly Thr His Ser Gln Trp Asn Lys 65 70 75 80 Pro
Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 85 90
95 Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala
100 105 110 Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu Asp
Arg Tyr 115 120 125 Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val
Tyr Tyr Arg Pro 130 135 140 Val Asp Gln Tyr Asn Asn Gln Asn Thr Phe
Val His Asp Cys Val Asn 145 150 155 160 Ile Thr Val Lys Gln His Thr
Val Thr Thr Thr Thr Lys Gly Glu Asn 165 170 175 Phe Thr Glu Thr Asp
Ile Lys Met Met Glu Arg Val Val Glu Gln Met 180 185 190 Cys Ile Thr
Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly 195 200 205 Ala
Ser 210 8240PRTMesocricetus auratus 8Met Trp Thr Asp Val Gly Leu
Cys Lys Lys Arg Pro Lys Pro Gly Gly 1 5 10 15 Trp Asn Thr Gly Gly
Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly 20 25 30 Asn Arg Tyr
Pro Pro Gln Gly Gly Gly Thr Trp Gly Gln Pro His Gly 35 40 45 Gly
Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 50 55
60 Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly
65 70 75 80 Thr His Asn Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn
Met Lys 85 90 95 His Met Ala Gly Ala Ala Ala Ala Gly Ala Val Val
Gly Gly Leu Gly 100 105 110 Gly Tyr Met Leu Gly Ser Ala Met Ser Arg
Pro Met Met His Phe Gly 115 120 125 Asn Asp Trp Glu Asp Arg Tyr Tyr
Arg Glu Asn Met Asn Arg Tyr Pro 130 135 140 Asn Gln Val Tyr Tyr Arg
Pro Val Asp Gln Tyr Asn Asn Gln Asn Asn 145 150 155 160 Phe Val His
Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr 165 170 175 Thr
Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys Ile Met 180 185
190 Glu Arg Val Val Glu Gln Met Cys Thr Thr Gln Tyr Gln Lys Glu Ser
195 200 205 Gln Ala Tyr Tyr Asp Gly Arg Arg Ser Ser Ala Val Leu Phe
Ser Ser 210 215 220 Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile Phe
Leu Met Val Gly 225 230 235 240 9254PRTMus musculus 9Met Ala Asn
Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr
Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25
30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg
35 40 45 Tyr Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly Gly
Gly Trp 50 55 60 Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His
Gly Gly Ser Trp 65 70 75 80 Gly Gln Pro His Gly Gly Gly Trp Gly Gln
Gly Gly Gly Thr His Asn 85 90 95 Gln Trp Asn Lys Pro Ser Lys Pro
Lys Thr Asn Leu Lys His Val Ala 100 105 110 Gly Ala Ala Ala Ala Gly
Ala Val Val Gly Gly Leu Gly Gly Tyr Met 115 120 125 Leu Gly Ser Ala
Met Ser Arg Pro Met Ile His Phe Gly Asn Asp Trp 130 135 140 Glu Asp
Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val 145 150 155
160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His
165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr
Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met
Met Glu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr Gln Tyr Gln
Lys Glu Ser Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser Ser Ser
Thr Val Leu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu Ile
Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 10253PRTHomo sapiens
10Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1
5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp
Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly
Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln
Pro His Gly Gly Gly 50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp
Gly Gln Pro His Gly Gly Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly
Gly Trp Gly Gln Gly Gly Gly Thr His 85 90 95 Ser Gln Trp Asn Lys
Pro Ser Lys Pro Lys Thr Asn Met Lys His Met 100 105 110 Ala Gly Ala
Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125 Met
Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp 130 135
140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln
145 150 155 160 Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn
Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His
Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr
Asp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile
Thr Gln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly
Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu
Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250 11253PRTHomo
sapiens 11Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala
Thr Trp 1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro
Gly Gly Trp Asn 20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly
Ser Pro Gly Gly Asn Arg 35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly
Trp Gly Gln Pro His Gly Gly Gly 50
55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly
Gly 65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly
Gly Thr His 85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr
Asn Met Lys His Met 100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val
Val Gly Gly Leu Gly Gly Tyr 115 120 125 Val Leu Gly Ser Ala Met Ser
Arg Pro Ile Ile His Phe Gly Ser Asp 130 135 140 Tyr Glu Asp Arg Tyr
Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr
Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val 165 170 175
His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr 180
185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu
Arg 195 200 205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu
Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe
Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile
Phe Leu Ile Val Gly 245 250
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