Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification

Article (PDF Available)inJournal of Biological Chemistry 281(46):35245-52 · December 2006with52 Reads
DOI: 10.1074/jbc.M603964200 · Source: PubMed
Abstract
Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

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Ultra-efficient Replication of Infectious Prions by Automated
Protein Misfolding Cyclic Amplification
*
S
Received for publication, April 25, 2006, and in revised form, August 21, 2006 Published, JBC Papers in Press, September 18, 2006, DOI 10.1074/jbc.M603964200
Paula Saa´
‡§
, Joaquı´n Castilla
, and Claudio Soto
‡1
From the
George and Cynthia Mitchell Center for Alzheimer Disease and Related Neurodegenerative Disorders, Departments of
Neurology, Neuroscience and Cell Biology, and Biochemistry and Molecular Biology, University of Texas Medical Branch,
Galveston, Texas 77555 and
§
Centro de Biologı´a Molecular, Universidad Auto´noma de Madrid, Madrid 28049, Spain
Prions are the unconventional infectious agents responsible
for transmissible spongiform encephalopathies, which appear
to be composed mainly or exclusively of the misfolded prion
protein (PrP
Sc
). Prion replication involves the conversion of the
normal prion protein (PrP
C
) into the misfolded isoform, cata-
lyzed by tiny quantities of PrP
Sc
present in the infectious mate-
rial. We have recently developed the protein misfolding cyclic
amplification (PMCA) technology to sustain the autocatalytic
replication of infectious prions in vitro. Here we show that
PMCA enables the specific and reproducible amplification of
exceptionally minute quantities of PrP
Sc
. Indeed, after seven
rounds of PMCA, we were able to generate large amounts of
PrP
Sc
starting from a 1 10
12
dilution of scrapie hamster
brain, which contains the equivalent of 26 molecules of pro-
tein monomers. According to recent data, this quantity is simi-
lar to the minimum number of molecules present in a single
particle of infectious PrP
Sc
, indicating that PMCA may enable
detection of as little as one oligomeric PrP
Sc
infectious particle.
Interestingly, the in vitro generated PrP
Sc
was infectious when
injected in wild-type hamsters, producing a disease identical to
the one generated by inoculation of the brain infectious mate-
rial. The unprecedented amplification efficiency of PMCA leads
to a several billion-fold increase of sensitivity for PrP
Sc
detec-
tion as compared with standard tests used to screen prion-
infected cattle and at least 4000 times more sensitivity than the
animal bioassay. Therefore, PMCA offers great promise for the
development of highly sensitive, specific, and early diagnosis of
transmissible spongiform encephalopathy and to further under-
stand the molecular basis of prion propagation.
Prion diseases or transmissible spongiform encephalopa-
thies (TSEs)
2
are neurodegenerative disorders of humans and
animals. Historically, scrapie has been the most common TSE
in animals, affecting sheep for over 200 years (1). The most
recent and worrisome outbreak of an animal TSE disease is
bovine spongiform encephalopathy (BSE) in cattle (2). BSE has
important implications for human health, because the infec-
tious agent can be transmitted to humans producing a new
disease, termed variant Creutzfeldt-Jakob disease (3, 4). TSEs
are characterized by an extremely long incubation period, fol-
lowed by a brief and invariably fatal clinical disease (5). To date
no therapy or early diagnosis is available.
The pathogen responsible for TSEs, called “prion,” is com-
prised mainly or exclusively of a misfolded protein named
PrP
Sc
, which is a post-translationally modified version of the
normal prion protein (PrP
C
) (6, 7). During the course of the
disease, prions replicate by the autocatalytic conversion of PrP
C
into PrP
Sc
, triggered by the misfolded protein present in the
infectious inoculum. The conversion seems to involve a confor-
mational change whereby the
-helical content of the normal
protein diminishes and the amount of
-sheet increases (8, 9).
The structural changes are accompanied by alterations in the
biochemical properties as follows: PrP
C
is soluble in nondena-
turing detergents; PrP
Sc
is insoluble; PrP
C
is readily digested by
proteases; and PrP
Sc
is partially resistant (7, 10).
To understand the mechanism of prion conversion, Caughey
and co-workers (11) developed a cell-free conversion reaction
that mimics in many aspects the prion replication process. One
of the limitations of the cell-free conversion method is the rel-
atively low efficiency of PrP
Sc
formation that diminishes its
application to study the nature of the infectious agent and to
attempt sensitive detection of the protein. More recently, we
have developed a novel technique referred to as protein mis-
folding cyclic amplification (PMCA), in which it is possible to
simulate prion replication in the test tube in an accelerated and
efficient way (12). In a cyclic manner, conceptually analogous to
PCR cycling, PrP
Sc
is incubated with excess PrP
C
to enlarge the
PrP
Sc
aggregates, which are then sonicated to generate multiple
smaller units for the continued formation of new PrP
Sc
(13). We
have reported previously proof-of-concept experiments in
which the technology was applied to replicate the misfolded
protein from diverse species (12, 14). The newly generated pro-
tein exhibits the same biochemical, biological, and structural
properties as brain-derived PrP
Sc
, and strikingly, it is infectious
to wild-type animals, producing a disease with characteristics
that are identical to the illness produced by brain-isolated pri-
ons (15). The technology has been automated, leading to a dra-
matic increase in efficiency of amplification and its application
* This work was supported in part by National Institutes of Health Grants
AG0224642 and NS049173. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must there-
fore be hereby marked advertisement in accordance with 18 U.S.C. Sec-
tion 1734 solely to indicate this fact.
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1.
1
To whom correspondence should be addressed: Dept. of Neurology, Uni-
versity of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555.
Tel.: 409-747-0017; Fax: 409-7470020; E-mail: clsoto@utmb.edu.
2
The abbreviations used are: TSE, transmissible spongiform encephalopathy;
PMCA, protein misfolding cyclic amplification; sa PMCA, serial automated
PMCA; BSE, bovine spongiform encephalopathy; PBS, phosphate-buffered
saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent
assay; PTA, phosphotungstic acid; PK, proteinase K.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35245–35252, November 17, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
NOVEMBER 17, 2006 VOLUME 281 NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35245
at The Scripps Research Institute on February 8, 2007 www.jbc.orgDownloaded from
http://www.jbc.org/cgi/content/full/M603964200/DC1
Supplemental Material can be found at:
to detect PrP
Sc
in blood of hamsters experimentally infected
with scrapie (16).
In this study we describe in detail the methodological aspects
for efficient PMCA amplification and the characterization of
the technology for reproducibility, specificity, and sensitivity.
We also study the minimum number of molecules needed to
trigger amplification and the application of the PMCA tech-
nique to generate infectious material in vitro. Our results dem-
onstrate that PMCA is capable of detecting as little as 26
monomers of PrP, which, according to recent data on the min-
imal size of the infectious particle (17), would correspond to a
single molecule of oligomeric infectious PrP
Sc
. The technique is
highly reproducible and specific to amplify PrP
Sc
, because no
signal is detected when no PrP
Sc
inoculum is present. Finally,
our results show that PMCA enables the increase of infectivity
by around 20 million-fold, converting a sample that is not infec-
tious into a highly infectious one. These data demonstrate that
PMCA has a similar power of amplification as PCR techniques
used to amplify DNA and have great promise for the develop-
ment of highly sensitive detection of PrP
Sc
and for understand-
ing the molecular basis of prion replication.
EXPERIMENTAL PROCEDURES
Preparation of Tissue Homogenates—Healthy and sick ani-
mals were perfused with phosphate-buffered saline (PBS) plus 5
m
M EDTA prior to harvesting the tissue. Ten percent brain
homogenates (w/v) were prepared in conversion buffer (PBS
containing 150 m
M NaCl, 1.0% Triton X-100, 4 m M EDTA, and
the Complete Protease Inhibitor Mixture from Roche Applied
Science). The samples were clarified by a brief, low speed cen-
trifugation (1500 rpm for 30 s) using an Eppendorf centrifuge,
model 5414 (Hamburg, Germany). Dilutions of this brain
homogenate were done in conversion buffer, and they are
expressed in relation to the brain; for example, a 100-fold dilu-
tion is equivalent to a 1% brain homogenate.
In Vivo Infectivity Studies—Syrian Golden hamsters were
used as an experimental model of scrapie. Animals were 4 6-
weeks old at the time of inoculation. Anesthetized animals were
injected intracerebrally stereotaxically in the right hippocam-
pus with 1
l of the sample or intraperitoneally with 200
lof
sample as described previously (15). The onset of clinical dis-
ease was measured by scoring the animals twice a week using
the following scale: 1, normal animal; 2, mild behavioral abnor-
malities, including hyperactivity and hypersensitivity to noise;
3, moderate behavioral problems, including tremor of the
head, ataxia, wobbling gait, head bobbing, irritability, and
aggressiveness; 4, severe behavioral abnormalities, including
all of the above plus jerks of the head and body and sponta-
neous backrolls; 5, terminal stage of the disease in which the
animal lies in the cage and is no longer able to stand up.
Animals scoring level 4 during 2 consecutive weeks were
considered sick and were sacrificed to avoid excessive pain
using exposition to carbonic dioxide. The scrapie infectious
material used in these studies was titrated and 1 LD
50
was
obtained in a brain dilution of 1 10
9
.
Brains were extracted and analyzed biochemically and histo-
logically. The right cerebral hemisphere was frozen and stored
at 70 °C for biochemical examination of PrP
res
using Western
blot analysis as described below. The left hemisphere was fixed
in 10% formaldehyde solution, cut into sections, and embedded
in paraffin. Serial sections (6
m thick) from each block were
stained with hematoxylin-eosin, using standard protocols, or
incubated with monoclonal antibodies recognizing PrP or the
glial fibrillary acidic protein. Immunoreactions were developed
using the peroxidase-antiperoxidase method, following the
manufacturer’s specifications. Antibody specificity was verified
by absorption. To compare the pattern of histopathological
damage among animals, we calculated the lesion profile, using a
variation of the method employed in mice (18). Briefly, the
severity of vacuolation was scored in a scale from 0 to 5 in seven
different brain areas, including medulla, cerebellum, superior
colliculus, hippocampus, and cerebral cortex occipital, frontal,
and lateral.
PMCA Procedure—Although the principle of PMCA remains
the same as in our original publication (12), the system has been
optimized and automated, thus enabling the routine processing
of many more samples in the same amount of time while reach-
ing a higher conversion efficiency. Aliquots of normal and
scrapie brain homogenate prepared in conversion buffer were
mixed and loaded onto 0.2-ml PCR tubes. Tubes were posi-
tioned on an adaptor placed on the plate holder of a
microsonicator (Misonix model 3000, Farmingdale, NY) and
programmed to perform cycles of 30 min of incubation at
37 °C followed by a 40-s pulse of sonication set at 60%
potency. Samples were incubated without shaking and
immersed in the water of the sonicator bath. and the entire
microplate horn was kept inside an incubator at 37 °C. The
detailed protocol, including troubleshooting, has been
recently published elsewhere (19–21).
Proteinase K Digestion—Samples were incubated with 50
g/ml PK for 60 min at 45 °C. The digestion was stopped by
adding electrophoresis sample buffer. In each experiment it is
important to have a negative control consisting of normal brain
homogenate and to check that PK digestion of PrP
C
is complete
to avoid confusion between undigested PrP
C
and the signal
from the PK-resistant core of PrP
Sc
. For this purpose special
care has to be taken to observe the switch in the molecular
weight after PK digestion, characteristic of bona fide PrP
Sc
.
Western Blot—Proteins were fractionated by SDS-PAGE
under reducing conditions, electroblotted into nitrocellulose
membrane, and probed with 3F4 antibody (22) (Signet,
Dedham, MA) diluted 1:5000 in PBS. The immunoreactive
bands were visualized by enhanced chemiluminescence assay
(Amersham Biosciences). Densitometric analysis was done by
using a UVP Bioimaging system EC3 apparatus (Upland, CA).
Statistical significance of the values was evaluated by Student’s
t test.
ELISA—Twenty
l of serial dilutions of an infected hamster
brain into 10% normal brain homogenate were treated with 50
g/ml PK for1hat45°Cand450rpm. The digestion was
stopped with 50 m
M phenylmethylsulfonyl fluoride. The
digested samples were mixed with PBS up to 100
l, loaded
onto a 96-well ELISA plate (Maxisorp surface treatment), and
incubated for1hat37°C.Theplates were blocked with 5% BSA
inPBSfor1hat3C,followed by incubation for 1 h with the
monoclonal antibody 3F4 (Signet, Dedham, MA) diluted 1:3000
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in 1% BSA in PBS. The antibody was washed four times with 200
l of PBS, 0.05% Tween 20, and samples were incubated with
the polyclonal antibody anti-mouse IgG, conjugated with
horseradish peroxidase (Amersham Biosciences), and diluted
1:1000 in 1% BSA in PBS. After four washes the samples were
developed with ImmunoPure ABTS (Pierce) following the
manufacturer’s directions.
PTA Precipitation—Twenty
l of serial dilutions of an
infected hamster brain into 10% normal brain homogenate
were first treated with benzonase (2.5 units/
linPBS,1mM
MgCl
2
) for 30 min at 37 °C with constant agitation. Thereafter,
we added 4% PTA (prepared in 70 m
M MgCl
2
, titrated with
NaOH to pH 7.4) and incubated at 37 °C for 30 min with con-
stant agitation. Samples were centrifuged at 15,800 g for 30
min at 37 °C in an Eppendorf microcentrifuge. Pellet was resus-
pended in 20 –30
l of PBS containing 0.1% Sarkosyl. After
treatment with PK, samples were analyzed by Western blot as
described above.
PrP
Sc
Quantitation—To estimate the quantity and the num-
ber of molecules of PrP
Sc
in our samples, we analyzed several
dilutions of scrapie brain homogenate by Western blot in the
same gel as aliquots of known amounts of recombinant hamster
PrP (supplemental Fig. 1 A). The signal intensity was evaluated
by densitometry, and the quantity of PrP in the sample was
estimated by extrapolation of the calibration curve prepared
with recombinant PrP (supplemental Fig. 1B). To minimize
artifacts because of saturated or weak signal, several different
dilutions were measured, and each dilution was analyzed in
triplicate. To standardize the signal among the different blots,
the densitometric data were expressed relative to the value of
the signal of the same quantity of normal brain homogenate
(without PK treatment). PrP
Sc
quantitation was also confirmed
by ELISA using recombinant PrP as standard. In this way we
estimated the average concentration of PrP
Sc
in the scrapie
brain used in our studies to be 67 ng/
l. The number of mole-
cules of PrP
Sc
detected was estimated by mathematical calcula -
tion of the dilution used and the known concentration of PrP
Sc
in the brain homogenate.
RESULTS
The PMCA Technology and Its Reproducibility—PMCA con-
sists of cycles of accelerated prion replication. The basis for
PMCA is the observation that prion replication follows a seed-
ing-nucleation model in which oligomeric PrP
Sc
in the infec-
tious material converts PrP
C
by integrating monomeric pro-
teins into the ends of the aggregate, inducing and stabilizing its
misfolding (13, 23). PMCA is a cyclic process consisting of two
phases. During the first phase the samples containing minute
amounts of PrP
Sc
and a large excess of PrP
C
were incubated to
induce growing of PrP
Sc
polymers. In the second phase the sam-
ple is sonicated to break down the polymers, multiplying the
number of nuclei. In this way, after each cycle the number of
“seeds” is increased in an exponential fashion. The number of
cycles can be repeated as many times as needed to reach the
amplification rate desired. For practical operation, the system
has been automated by using a programmable plate sonicator,
as described under “Experimental Procedures.” This improve-
ment not only decreases processing time and allows for the
routine processing of many more cycles than a single probe
sonicator but also prevents loss of material. Cross-contamina-
tion is eliminated because there is no direct probe intrusion
into the sample.
Reproducibility of amplification was measured by monitor-
ing the PrP
Sc
signal obtained before and after PMCA cycling
under different experimental conditions. Equivalent samples
containing a 10,000-fold dilution of scrapie brain into 10%
healthy hamster brain homogenate were placed in distinct posi-
tions of the microplate sonicator and subjected to 48 PMCA
cycles. The levels of amplification were studied by Western blot
after PK digestion (Fig. 1A), and data were quantitated by den-
sitometric analysis of the Western blot signal (Fig. 1B).
Although some small variability was observed on the signal
obtained in distinct wells, the differences were not significant
and could not be attributed to a position effect, but rather they
probably reflect some small experimental variability.
To analyze further the reproducibility of the procedure,
equivalent samples containing a 10,000-fold dilution of scrapie
brain homogenate into 10% healthy hamster brain homogenate
were subjected to 48 PMCA cycles in experiments done on
different days. Fig. 1C shows that at 7 distinct days the amplifi-
cation efficiency was virtually the same. Again, densitometric
analysis showed that the signal was not statistically different in
the distinct experiments (Fig. 1D). For this experiment, the nor-
mal brain sample used on day 1 was freshly prepared, whereas
for all the other days, frozen material was used. Therefore, no
differences between fresh and frozen material were observed.
However, it is important to note that brain substrate has to be
frozen in aliquots to avoid repetitive freezing-thawing that
decreases PMCA efficiency.
The influence of different, but equivalent, inocula on the
conversion efficiency was studied by amplifying preparations of
10,000-fold diluted scrapie brain homogenate obtained from
five distinct hamsters into the same substrate (Fig. 1E). After 48
PMCA cycles, a large and similar conversion of PrP
C
into PrP
Sc
was observed. A similar result was obtained when normal brain
homogenate from five different hamsters was used as a sub-
strate for the amplification of a unique PrP
Sc
inoculum (Fig.
1G). However, densitometric analysis of the experiments
shown in Fig. 1, E and G, indicate that in both cases one sample
gave a statistically significant different level of amplification
than the other four samples (Fig. 1, F and H, respectively).
These differences are not because of changes in the levels of
PrP
Sc
or PrP
C
in the samples, which were not significantly dif-
ferent. These results suggest that perhaps individual variability
on the expression of PrP or conversion factors may lead to
changes on the extent of prion conversion in vitro. In none of
the experiments shown in Fig. 1 was PrP
Sc
detectable in samples
containing the same material but kept frozen without
amplification.
Specificity of PMCA Amplification—Specificity of cyclic
amplification was evaluated in a blind study in which 10 brain
samples of scrapie-affected hamsters and 11 samples of healthy
animals were subjected to 48 PMCA cycles, and PrP
Sc
was
detected by Western blot analysis after PK digestion (Fig. 2, A
and B). The results show that although 100% of the samples
derived from sick animals (Fig. 2, A and B, lanes 1, 2, 3, 5, 7, 11,
In Vitro Replication of Infectious Prions
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13, 17, 19, and 21) were positive after PMCA, none of the sam-
ples coming from normal animals (lanes 4, 6, 8, 9, 10, 12, 14, 15,
16, 18, and 20) show any significant PrP
Sc
signal. Of the 10
positive control samples, 7 corresponded to a 10,000-fold dilu-
tion of brain, 2 corresponded to a 50,000-fold dilution (Fig. 2A,
lanes 13 and 17 in), and 1 corresponded to a 100,000-fold dilu-
tion (Fig. 2A, lane 19). None of these 10 samples showed any
PrP
Sc
signal in Western blot without PMCA amplification (data
not shown). The interpretation of the data is that, under the
conditions used, PMCA leads to 100% specificity for PrP
Sc
detection.
As demonstrated before, the amplification rate using PMCA
depends upon the number of incubation/sonication cycles car-
ried out (12). Thus, we decided to evaluate whether a PrP
Sc
-like
signal might appear on negative samples after many PMCA
cycles. For this purpose, a 10% healthy hamster brain homoge-
nate in the absence (negative control) or in the presence (posi-
tive control) of an aliquot of a 50,000-fold diluted scrapie brain
was subjected to 24, 48, 96, or 144 PMCA cycles, and the PrP
Sc
signal was detected by Western blot analysis (Fig. 2C). The
results clearly indicate that PrP
Sc
reactivity was detected only
after PMCA in the positive control samples with an intensity
that depended upon the number of cycles performed. In com-
parison, in the negative control samples, no PrP
Sc
was ever
detected, regardless of the number of PMCA cycles carried out
(Fig. 2C).
Specificity was further studied in an even more challenging
situation in which several rounds of PMCA were done after
FIGURE 1. Reproducibility of automated PMCA. A, samples containing a 10,000-fold dilution of 263K scrapie hamster brain prepared in 10% normal hamster
brain were either immediately frozen (F) or placed in 10 different positions of the plate holder and subjected to 48 PMCA cycles. Thereafter, samples were
treated with PK and PrP
Sc
reactivity analyzed by Western blotting. B, densitometric analysis of three different blots obtained using the samples described in A.
C, equivalent samples prepared as described in A were subjected to 48 PMCA cycles performed at seven different times, and the extent of PrP
Sc
formation was
evaluated by Western blotting. D, densitometric analysis of three different blots obtained using the samples described in C. E, five different scrapie hamster
brains (I1 through I5) were diluted 10,000-fold into the same 10% normal hamster brain homogenate and subjected to 48 PMCA cycles. PrP
Sc
signal was
detected by Western blotting after PK treatment. F, densitometric analysis of three different blots obtained using the samples described in E. G, a single scrapie
brain homogenate was diluted 10,000-fold into 10% solutions prepared from five different normal hamster brains (S1 through S5). Again 48 PMCA cycles were
performed and PrP
Sc
detected by Western blotting. H, densitometric analysis of three different blots obtained using the samples described in G. F, frozen
samples; A, amplified samples. NBH, normal brain homogenate. All samples were treated with PK before electrophoresis, except those in which PK is
indicated. Data were statistically analyzed by one-way analysis of variance and Student’s t test. * p 0.05.
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diluting the material to refresh the substrate. We have shown
that PrP
Sc
can be kept replicating indefinitely in vitro by several
rounds of successive PMCA (15) and that serial PMCA enables
the ultrasensitive detection of PrP
Sc
in brain and blood samples
(16). Brains from healthy hamsters and from animals infected
with the 263K scrapie strain were diluted 10
4
-fold into a 10%
normal hamster brain homogenate. Samples were subjected to
48 PMCA cycles. After this first round of PMCA, a small aliquot
of the amplified samples was taken and diluted 10-fold into
more normal brain homogenate. These samples were again
amplified by 48 PMCA cycles. This procedure was repeated
several times, and PrP
Sc
generation was determined by Western
blot analysis after PK digestion. As shown in Fig. 2D, 10 rounds
of PMCA to reach a final dilution of the original brain equiva-
lent to 10
13
led to continuous formation of PrP
Sc
only when
the initial inoculum was derived from scrapie-infected animals.
No PrP
Sc
was ever detected in the absence of PrP
Sc
inoculum,
indicating that the system retains high specificity, even after
480 PMCA cycles. These same samples were used for further
amplification, and after a dilution of more than 10
63
of initial
inoculum a robust and continuous amplification was observed
in samples containing PrP
Sc
. When many additional rounds of
saPMCA were performed, we observed a scatter appearance of
a protease-resistant band identical to PrP
Sc
even in samples
without PrP
Sc
inoculum (data not shown). Spontaneous gener -
ation of PrP
Sc
was seen mainly when
more than 10 rounds of PMCA were
done. At present we do not know
whether this newly generated PrP
Sc
is the result of cross-contamination
or the de novo generation of PrP
Sc
.
More experiments need to be per-
formed to analyze the reproducibil-
ity of this observation and to dis-
tinguish between these two possibil-
ities. In case we ruled out the possi-
bility of contamination, these find-
ings would indicate that PMCA can
amplify a low frequency event in
which PrP
C
spontaneously converts
into PrP
Sc
. This could be the basis
for the origin of sporadic prion
diseases.
Sensitivity of PMCA and Mini-
mum Number of Molecules Detected
after Amplification—We have re-
cently reported that sensitivity of
detection after 140 cycles of PMCA
was increased by around 6600-fold
(16). We also reported previously
that to further increase replication
efficiency, we needed to refresh the
substrate periodically using a meth-
odology we termed serial auto-
mated PMCA (saPMCA) (16). By
performing two rounds of saPMCA,
we were able to detect PrP
Sc
up to a
5 10
10
dilution of scrapie ham-
ster brain, to reach an increase of sensitivity of around 10 mil-
lion-fold (16). To estimate the minimum number of molecules
of PrP
Sc
that our technology can detect in a given sample, we
diluted a scrapie brain homogenate 1 10
12
-fold into conver-
sion buffer and subjected this material to saPMCA. According
to our estimations, the PrP
Sc
concentration in the scrapie-in-
fected brain used for these studies was 67 ng/
l (see “Exper-
imental Procedures” and supplemental Fig. 1). This result indi-
cates that a 1 10
12
-fold dilution should contain 6.7
10
20
g/
l or 1.3 molecules of PrP
Sc
monomer per
l. Because
in our experiments we use a volume of 20
l, the sample tested
contains 26 molecules of monomeric PrP
Sc
. Strikingly, after
five rounds of saPMCA, we were able to detect a signal in one of
the four replicates used, and after seven rounds of amplifica-
tion, we detected a signal in three of the four replicates (Fig. 3).
Importantly, no amplified product was detected when a 10
14
-
fold dilution of brain was used (a sample that should contain no
molecules of PrP
Sc
) or in any of the control samples in which no
PrP
Sc
was present (Fig. 3). No signal was detected either in a
10
13
-fold dilution (data not shown).
The serious consequences of the BSE epidemics and the
increasing concern regarding the iatrogenic transmission of
variant Creutzfeldt-Jakob disease have motivated the develop-
ment of several biochemical methods to detect PrP
Sc
. Several
tests have been approved by the European community and are
FIGURE 2. Specificity of PrP
Sc
detection after PMCA. A, blind study in which aliquots from 11 different normal
brain samples and 10 distinct diluted scrapie-infected brain samples were subjected to 48 PMCA cycles to
attempt detection of PrP
Sc
. For all scrapie-infected samples, except for the ones in lanes 13, 17, and 19, the brain
was diluted 10,000-fold into 10% normal brain homogenate. For lanes 13 and 17, the inoculum was diluted
50,000 and for lane 19, the scrapie brain was diluted 100,000-fold. After amplification, the samples were treated
with PK and analyzed by Western blotting. B, densitometric analysis of three different blots from the samples
shown in A. C, samples from healthy (denoted PrP
Sc
inoculum) or scrapie-infected (denoted PrP
Sc
inoculum)
brain were diluted 50,000 into 10% normal brain homogenate and subjected to 0, 24, 48, 96, or 144 PMCA
cycles, and PrP
Sc
signal was analyzed by Western blotting. D, brains from healthy hamsters or from animals
infected with 263K scrapie were diluted 10
4
-fold into a 10% normal hamster brain homogenate. Samples were
subjected to 48 PMCA cycles. The amplified material was diluted 10-fold into normal brain homogenate and
amplified again. This procedure was repeated several times to reach a 10
13
dilution of initial material. The
figure shows the PrP
Sc
signal obtained in Western blots of only some of the dilutions, representative of all the
results obtained. NBH, normal brain homogenate. All samples were treated with PK before electrophoresis,
except those in which PK is indicated.
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widely used in BSE surveillance in several countries (24, 25). All
these tests consist of the immunological detection of PrP
Sc
either by Western blot (26), ELISA (27), or the conformational
dependent immunoassay (28). To compare the sensitivity of
these standard tests with PMCA detection, we performed stud-
ies in parallel with different methods using the same samples,
all prepared in 10% normal brain homogenate to facilitate the
comparison (Table 1). Western blot is the most standard but
least sensitive of these tests, allowing for the detection of a min-
imum of 4.0 ng of PrP
Sc
in 20
l of sample, which is equivalent
to 8 10
10
molecules of misfolded protein. Simple ELISA was
8-fold more sensitive than Western blotting (Table 1). One
strategy that has been used to enhance PrP
Sc
detection is the
specific precipitation and concentration of the protein using
PTA (29, 30). Under our experimental conditions, PTA precip-
itation of PrP
Sc
from scrapie brain led to a 50-fold increase in
detection (Table 1). According to literature reports, conforma-
tional dependent immunoassay detects PrP
Sc
up to a maximum
dilution of hamster scrapie brain homogenate equivalent to 5
10
5
(29), indicating that it is 60-fold more sensitive than West-
ern blotting (Table 1). By comparison, one round of 100 PMCA
cycles resulted in an average of 2500-fold more sensitive detec-
tion of PrP
Sc
as compared with Western blotting. This sensitiv-
ity threshold indicates that one round of PMCA can detect as
little as 3.2 10
7
molecules of PrP
Sc
in a 20-
l sample volume
(Table 1). On the other hand, two rounds of 100 PMCA cycles
were able to systematically detect PrP
Sc
up to a maximum dilu-
tion of the scrapie brain equivalent to 5 10
10
, indicating a
sensitivity 6 million times higher than standard Western blot-
ting (Table 1). In other words, two rounds of 100 PMCA cycles
can detect as little as 13,000 molecules of PrP
Sc
. As shown
before, seven successive rounds of PMCA cycles produce a sig-
nal after amplification even when the starting material was a
1 10
12
-fold dilution of sick brain homogenate. This ampli-
fication leads to an increase of sensitivity of 3 billion times with
respect to standard Western blot (Table 1). Until now, the ani-
mal bioassay of infectivity was by far the most sensitive assay
available for detection of prions (31, 32). Among the animal
bioassays, hamsters infected with the 263K scrapie strain are
the most rapid and sensitive, because animals can be infected
with the lowest quantity of infectious agent, and disease symp-
toms are observed at the shortest time after inoculation (33).
Indeed, a 1 10
9
dilution of sick brain is the minimum
amount that can still produce disease in 50% of the animals
(mean lethal dose or LD
50
). In our experiments the minimum
dilution that produced disease in all animals was 4 10
9
,
indicating that the bioassay can detect as little as 107,000 mol-
ecules of misfolded protein, which represent a 725,000-fold
higher sensitivity than Western blotting (Table 1). Remarkably,
our findings with saPMCA using the same samples as for the
FIGURE 3. Minimum quantity of PrP
Sc
detected by saPMCA. Aliquots of
scrapie hamster brain homogenate were serially diluted into conversion
buffer to reach 1 10
12
and 1 10
14
dilutions. Four aliquots of 20
lof
each dilution were mixed in four separated tubes with 80
l of normal brain
homogenate and subjected to 144 PMCA cycles. Thereafter, a volume of 20
l
was used for Western blotting after PK digestion, and 10
l were diluted into
90
l of normal brain homogenate, and the samples were subjected to a
second round of 144 PMCA cycles. The procedure was repeated several times
to reach seven successive rounds of PMCA. S1, S2, S3, and S4 correspond to the
four replicated tubes in each dilution. As a negative control, we used normal
brain homogenate diluted 10
12
into conversion buffer and subjected to the
same scheme of saPMCA. This experiment was also done in four replicated
tubes, and C1, C2, C3, and C4 represent each result.
TABLE 1
Comparison of the sensitivity of several methods to detect PrP
Sc
Assay
Maximum dilution
detected
a
Minimum PrP
quantity detected
b
Minimum no. of
PrP molecules
c
Increase in
sensitivity
d
Standard Western blot 3.0 10
3
4.0 ng 8.0 10
10
1
ELISA 3.7 10
4
0.5 ng 1.0 10
10
8
Phosphotunstic acid precipitation 6.0 10
5
80 pg 1.6 10
9
50
Conformation dependent immunoassay
e
5.0 10
5
67 pg 1.3 10
9
60
Animal bioassay 2.0 10
9
5.3 fg 1.1 10
5
725,000
One round of PMCA
f
1.2 10
6
1.6 pg 3.2 10
7
2,500
Two rounds of PMCA
f
5.0 10
10
0.7 fg 1.3 10
4
6,000,000
Seven rounds of PMCA
f
1.0 10
12
1.3 ag 26 3,000,000,000
a
The maximum dilution detected corresponds to the last dilution of 263K scrapie brain in which PrP
Sc
is detectable.
b
The minimum quantity of PrP
Sc
detected in a brain sample volume of 20
l.
c
The number of PrP molecules detected in a 20
l sample volume was estimated as described in supplemental Fig. 1 by comparison with recombinant PrP.
d
The increase of sensitivity is expressed in relation to the standard Western blot assay using 3F4 antibody.
e
The data for the conformation-dependent immunoassay were taken from the literature, whereas all the others were experimentally calculated.
f
The data for PMCA correspond to the average obtained in three different experiments using 100 PMCA cycles in each round.
In Vitro Replication of Infectious Prions
35250 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 46 NOVEMBER 17, 2006
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infectivity studies demonstrate that two and seven rounds of
saPMCA are 8 and 4000 times more sensitive than the most
efficient animal bioassay, respectively (Table 1).
Generation of Infectivity in Vitro from Sub-infectious Quan-
tities of PrP
Sc
—As described in Fig. 3, we were able to generate
large quantities of PrP
Sc
from a very high dilution (1 10
12
)of
hamster scrapie brain. Mathematical calculation estimates that
such dilution contains the equivalent of 26 molecules of PrP
monomers, which are at least 3– 4 orders of magnitude below
the minimum quantity of PrP
Sc
needed to produce infectivity in
some animals at very long times after inoculation. To study
whether the in vitro generation of PrP
Sc
was associated with an
increase of infectivity, we inoculated intraperitoneally the sam-
ple obtained after seven rounds of amplification starting with
1 10
12
dilution (Fig. 3, sample S
1
). All six wild-type hamsters
inoculated showed typical signs of 263K scrapie disease and
were sacrificed at an average of 299.6 20.5 days post-inocu-
lation. In our experience with this animal model, this incuba-
tion time is similar to that obtained with a 5 10
4
dilution of
scrapie infected brain inoculated by the intraperitoneal route.
Therefore, seven rounds of PMCA produced a 2 10
7
increase
of infectivity. As expected none of the control animals inocu-
lated with the same dilution without amplification developed
the disease even after 500 days post-inoculation.
The clinical signs observed in animals inoculated with the in
vitro amplified samples were identical to those of the animals
treated with infectious brain material and included hyperactiv-
ity, motor impairments, head wobbling, muscle weakness, and
weight loss. Brain samples from these animals contain a large
quantity of protease-resistant PrP
Sc
, which has an identical gly-
cosylation profile to the protein observed in brain-inoculated
animals (Fig. 4A). Conversely, no protease-resistant protein
was detected in the brain of negative control animals. Histolog-
ical analysis showed typical brain spongiform degeneration,
PrP accumulation, and astrogliosis (Fig. 4B). No differences in
the lesion pattern profile were observed compared with animals
inoculated by brain infectious material. These findings suggest
that PrP
Sc
generated in vitro corresponds to the same strain of
263K used to trigger amplification.
DISCUSSION
Our results indicate that PMCA is able to amplify exception-
ally small quantities of PrP
Sc
in a very specific and reproducible
manner. Indeed, by using several successive rounds of PMCA,
our data demonstrate that we are able to induce the conversion
of PrP
C
with as little as the equivalent to 26 molecules of PrP
Sc
monomers. Recent data have shown that the minimum size of
the particle able to sustain infectivity and to induce the cell-free
conversion of PrP
C
into PrP
Sc
contains between 14 and 28 mol-
ecules of PrP monomers (17). Taken together, this suggests that
saPMCA can amplify a single particle of oligomeric infectious
PrP
Sc
. This unprecedented amplification efficiency is compara-
ble only to the effectiveness of PCR amplification of DNA (34).
Moreover, although at these levels of amplification PCR often
results in artifactual amplification products, we rarely see a
false-positive using PMCA.
The applications of such a powerful amplification technology
are many and impact various fields. A particularly important
application is on the development of a highly sensitive bio-
chemical detection of PrP
Sc
, which constitutes the best surro-
gate marker for TSE diagnosis (24). A comparison of saPMCA
with the sensitivity of some of the current methodologies used
for PrP
Sc
detection (Table 1) showed that our technology is
several millions or even billions of times more sensitive than the
standard Western blot or ELISA-based assays currently used to
detect prions in cattle. At present, it is not possible to diagnose
the disease in the pre-clinical phase or in live individuals using
body biological fluids (24). The successful implementation of
saPMCA should lead to the identification of individuals and
tissue samples infected with even the tiniest quantities of PrP
Sc
,
enabling us to decrease the risk of further propagation of TSE to
the minimum. Indeed, we have recently reported for the first
time the biochemical detection of PrP
Sc
in blood samples using
saPMCA both at the symptomatic (16) and pre-symptomatic
stages of the disease (35). Although technically more challeng-
ing, the PMCA technology has been adapted to amplify prions
from a variety of origins, including human (14). One of the
major limitations in the case of human samples is the availabil-
ity of normal brain tissue to use as a substrate of PMCA, but this
can be overcome by using transgenic mice brain or cell lysates.
Another important application of PMCA is to understand
the underlying biology of prions and the nature of the infectious
agent. In this study we show that it is possible to generate infec-
tivity by amplification of a scrapie brain sample diluted 10
12
-
fold, containing an estimated single particle of oligomeric
PrP
Sc
. By successive in vitro replication of this single particle of
PrP
Sc
, we have successfully generated many millions of PrP
Sc
FIGURE 4. Biochemical and histological features of the disease induced
by inoculation with in vitro generated PrP
Sc
. A, samples of brain homoge-
nate from animals inoculated with PrP
Sc
amplified from a 1 10
12
dilution
of scrapie brain (line 4), with brain infectious agent (line 3), or with control
normal brain homogenate (line 2) were treated with 50
g/ml PK for 60 min at
45 °C. PrP signal was detected by Western blot using the 3F4 antibody. Line 1
corresponds to the control normal brain homogenate without PK treatment.
B, histological evaluation of brain sections from an animal inoculated with in
vitro amplified PrP
Sc
showed the typical spongiform brain degeneration after
staining with hematoxylin-eosin (left panel), PrP accumulation as evaluated
by staining with the 3F4 anti-PrP monoclonal antibody (center panel), and
reactive astrogliosis as detected by staining with glial fibrillary acidic protein
antibodies (right panel). Similar results were obtained with several other ani-
mals inoculated with the same material. The profile of the brain lesion pattern
was indistinguishable from those obtained by inoculation of brain infectious
material.
In Vitro Replication of Infectious Prions
NOVEMBER 17, 2006 VOLUME 281 NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35251
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molecules and raised infectivity by more than 7 orders of mag-
nitude. These data support and extend our previous findings
showing the in vitro generation of prion-infectious material by
cell-free replication of PrP
Sc
using PMCA (15). These findings
provide strong support for the prion hypothesis. However, we
cannot rule out that other molecules present in the brain homo-
genate may also contribute to infectivity.
In addition, PMCA may be useful for understanding the
molecular mechanism of prion replication and the identifica-
tion of endogenous factors modulating PrP
Sc
formation.
Indeed, Supattapone and co-workers (36–39) have used PMCA
to show that metal cations, such as copper and zinc, and polya-
nions, including diverse types of RNA molecules, can modulate
PrP conversion in vitro. PMCA may also be used to examine
and quantify the species barrier phenomenon and to under-
stand the mechanism encoding prion strains. Finally, PMCA
may be used as an efficient high throughput screening assay for
identification of molecules able to inhibit or reverse prion rep-
lication and thus to discover novel potential drugs for TSE
treatment. An efficient treatment to stop further prion conver-
sion coupled with an early pre-symptomatic diagnosis to iden-
tify patients before irreversible brain damage has occurred
seems the most promising approach to combat these devastat-
ing diseases.
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In Vitro Replication of Infectious Prions
35252 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 46 NOVEMBER 17, 2006
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    • "Prion protein misfolding activity in PBMC isolated from recipient sheep was assessed by sPMCA as described previously but with some modifications [12, 13, 26]. PBMC isolated from inoculated and uninoculated sheep in group 2 at 15 mpi and group 3 at 17 mpi were used for analysis. "
    [Show abstract] [Hide abstract] ABSTRACT: Classical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes. PBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients. These findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.
    Full-text · Article · Dec 2016
    • "In the last decade various laboratories reported the detection of infectivity and PrP TSE in blood from animals with natural and experimental TSEs [15][16][17][18][19][20][21]41,69] and in blood of humans infected with vCJD and sCJD [5][6][7][8][9]27] . We previously presented data on the detection of PrP TSE by PMCA in EVs containing exosomes that were isolated from plasma of mice infected with movCJD [42] . "
    [Show abstract] [Hide abstract] ABSTRACT: Blood has been shown to contain disease-associated misfolded prion protein (PrPTSE) in animals naturally and experimentally infected with various transmissible spongiform encephalopathy (TSE) agents, and in humans infected with variant Creutzfeldt-Jakob disease (vCJD). Recently, we have demonstrated PrPTSE in extracellular vesicle preparations (EVs) containing exosomes from plasma of mice infected with mouse-adapted vCJD by Protein Misfolding Cyclic Amplification (PMCA). Here we report the detection of PrPTSE by PMCA in EVs from plasma of mice infected with Fukuoka-1 (FU), an isolate from a Gerstmann-Sträussler-Scheinker disease patient. We used Tga20 transgenic mice that over-express mouse cellular prion protein, to assay by intracranial injections the level of infectivity in a FU-infected brain homogenate from wildtype mice (FU-BH), and in blood cellular components (BCC), consisting of red blood cells, white blood cells and platelets, plasma EVs, and plasma EVs subjected to multiple rounds of PMCA. Only FU-BH and plasma EVs from FU-infected mice subjected to PMCA that contained PrPTSE transmitted disease to Tga20 mice. Plasma EVs not subjected to PMCA and BCC from FU-infected mice failed to transmit disease. These findings confirm the high sensitivity of PMCA for PrPTSE detection in plasma EVs and the efficiency of this in vitro method to produce highly infectious prions. The results of our study encourage further research to define the role of EVs and, more specifically exosomes, as blood-borne carriers of PrPTSE.
    Article · Jul 2016
    • "Although occasionally debated [43, 44], a large body of evidence indicates a direct correlation between infectivity and the PMCA seeding activity [36, 37, 39,45464748 . PMCA was also demonstrated as more sensitive than bioassays by several logs of magnitude for the detection of prions [36, 46, 49] . This extended limit of detection might originate from an absence of prion clearance mechanisms in PMCA reactions [50]. "
    [Show abstract] [Hide abstract] ABSTRACT: The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.
    Full-text · Article · Jan 2016
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