##################### Bovine Spongiform Encephalopathy #####################
From: TSS ()
Subject: Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy
Date: October 9, 2005 at 9:15 am PST
BMC Biotechnology
This Provisional PDF corresponds to the article as it appeared upon acceptance. The fully-formatted
PDF version will become available shortly after the date of publication, from the URL listed below.
Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood
from cattle affected with bovine spongiform encephalopathy
BMC Biotechnology 2005, 5:26 doi:10.1186/1472-6750-5-26
Lothar Trieschmann ([email protected].)
Alexander Navarrete Santos ([email protected].)
Katja Kaschig ([email protected].)
Sandra Torkler ([email protected].)
Elke Maas ([email protected])
Hermann Schatzl ([email protected])
Gerald Bohm ([email protected].)
ISSN 1472-6750
Article type Research article
Submission date 3 May 2005
Acceptance date 4 Oct 2005
Publication date 4 Oct 2005
Article URL http://www.biomedcentral.com/content/5/1/26
Like all articles in BMC journals, this peer-reviewed article was published immediately upon acceptance. It
can be downloaded, printed and distributed freely for any purposes (see copyright notice below).
Articles in BMC journals are listed in PubMed and archived at PubMed Central.
For information about publishing your research in BMC journals or any BioMed Central journal, go to
http://www.biomedcentral.com/info/authors/
© 2005 Trieschmann et al., licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
- 1 -
Ultra-sensitive detection of prion protein fibrils by
flow cytometry in blood from cattle affected with
bovine spongiform encephalopathy
Lothar Trieschmann1, Alexander Navarrete Santos1, Katja Kaschig1, Sandra Torkler1,
Elke Maas2, Hermann Schätzl2, Gerald Böhm1§
1ACGT ProGenomics AG, Weinbergweg 22, D-06120 Halle (Saale), Germany
2Institute of Virology, Technical University of Munich, Biedersteinerstrasse 29, D-
80802 Munich, Germany
§Corresponding author
Email addresses:
LT: [email protected].
ANS: [email protected].
KK: [email protected].
ST: [email protected].
HS: [email protected]
EM: [email protected]
GB: [email protected].
- 2 -
Abstract
Background
The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in
humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the
post mortem detection of the pathological form of the prion protein (PrPSc) in brain
tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even
at presymptomatic stages of the disease, albeit at concentrations well below the
detection limits of currently available analytical methods.
Results
We developed a highly sensitive method for detecting prion protein aggregates that
takes advantage of kinetic differences between seeded and unseeded polymerization
of prion protein monomers. Detection of the aggregates was carried out by flow
cytometry. In the presence of prion seeds, the association of labelled recombinant PrP
monomers in plasma and serum proceeds much more efficiently than in the absence
of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down
to a concentration of approximately 10-8 nM [0.24 fg/ml]. A specific signal was
detected in six out of six available serum samples from BSE-positive cattle.
Conclusions
We have developed a method based on seed-dependent PrP fibril formation that
shows promising results in differentiating a small number of BSE-positive serum
samples from healthy controls. This method may provide the basis for an ante mortem
diagnostic test for prion diseases.
- 3 -
Background
A group of fatal transmissible neurodegenerative diseases, including Creutzfeld-Jakob
disease (CJD), bovine spongiform encephalopathy (BSE), chronic wasting disease
(CWD) and scrapie, is caused by an unusual infectious agent that has been termed
prion [1]. Prions consist of an aberrant isoform (PrPSc) of the normal cellular prion
protein (PrPC). PrPC is a cell surface glycoprotein expressed in neurons [2] and other
cell types [3, 4]. The precise physiological function of the cellular prion protein is not
known yet. PrPSc differs from PrPC in its higher content of -sheet structure [5, 6], its
partial resistance to protease digestion [7], and its tendency to form large aggregates
[8]. PrPSc propagates by converting the cellular prion protein to the PrPSc
conformation [9]. PrPSc aggregates accumulate predominantly in the central nervous
system (CNS), and definitive diagnosis of prion diseases currently relies on the post
mortem detection of PrPSc in CNS tissue by immunohistochemistry, Western blotting,
or ELISA [10]. Transmission studies indicate that prions may also be present in
blood, potentially allowing for ante mortem diagnosis, but the sensitivity of the
currently available analytical methods is insufficient for the detection of the extremely
low prion titers that can be expected in body fluids [11].
Here, we report the development of a method based on kinetic differences between
seeded and unseeded aggregation of prion protein that allows the detection of PrP
aggregates in blood down to attomolar concentrations by flow cytometry.
Results and discussion
Detection of synthetic prion protein aggregates in serum or plasma
Kinetic differences between seeded and spontaneous polymerization of peptide
monomers can be used for the detection of amyloid -protein aggregates in the
- 4 -
cerebrospinal fluid of Alzheimer's disease patients [15]. Here, we extend the principle
of seeded polymerization to the detection of prion protein aggregates.
While trying to establish conditions for the labeling of synthetic prion protein
aggregates with a fluorescently labeled prion protein probe, we observed that the
formation of prion protein aggregates proceeds much less efficiently in serum or
plasma (not shown) than in PBS (Fig. 1). This inhibition is probably caused by
interactions of the prion protein probe with serum proteins.
Next, we found that the addition of preformed prion protein aggregates to plasma can
partially overcome this inhibition (Fig. 2). The preformed aggregates presumably
function as seeds that facilitate the formation of new aggregates in the inhibitory
environment of plasma. The seeds stimulated the formation of prion protein
aggregates at all concentrations tested, from 5 nM [120 ng/ml] to 10-8 nM [0.24 fg/ml]
(Fig 2C). The average ratio of event counts in seeded samples to those in samples
without seeds was 6.4. The number of events, however, was not proportional to the
seed concentration, but remained relatively constant over the whole concentration
range. Thus, the seed-dependent formation of prion protein aggregates can be used to
detect extremely low amounts (down to the attomolar range) of spiked prion protein
aggregates in blood.
Analysis of serum from clinical-stage, BSE-positive cattle
Studies demonstrating the transmission of prion diseases by blood transfusion suggest
that prions are present in the blood of afflicted animals and people, even at presymptomatic
stages of the disease [16, 17, 18]. We used the method of seeddependent
fibril formation to analyze serum from six confirmed cases of clinicalstage,
BSE-positive cattle and four controls. Based on the spiking experiments
described above, our hypothesis was that any PrPSc aggregates present in serum may
- 5 -
act as seeds for the formation of easily detectable amounts of labeled PrP aggregates,
whereas in the absence of seeds the formation of PrP aggregates would be inhibited.
The serum samples from BSE-positive cattle and controls from healthy cattle were
incubated with 10 nM of a FITC-labeled bovine PrP probe at 37°C for 20 h with
continuous shaking, followed by analysis in a flow cytometer. All six BSE-samples
could be clearly distinguished by a population of events that was absent in the
controls (Fig. 3A-J, green dots in region R3; quantification in fig. 3K).
Conclusions
We have developed a method based on seed-dependent PrP fibril formation that
shows promising results in differentiating a small number of BSE-positive serum
samples from healthy controls. More samples need to be tested in order to validate its
potential as an ante mortem diagnostic test for BSE and other prion diseases.
Methods
Biological fluids
Serum samples from six confirmed cases of BSE in cattle and four control animals
were obtained from BFAV, Insel Riems, Germany. Control plasma was obtained from
a blood bank.
Labeling of prion protein
Recombinant full-length bovine PrP was produced as described previously [12, 13].
The purified protein was labeled with a FITC-labeling kit (Roche) according to the
manufacturer's instructions.
- 6 -
Preparation of fibrils from recombinant prion protein
25 µM of unlabeled bovine prion protein in PBS containing 0.2 % SDS was incubated
for 10 min at room temperature, followed by a twentyfold dilution with PBS. For
fibril formation, the diluted reaction mixture was incubated for 48 h at room
temperature [14].
PrP fibril formation in serum or plasma
Recombinant FITC-labeled bovine prion protein was incubated in 150 µl serum or
plasma at a concentration of 5 or 10 nM for 5-10 min. at 20°C, shaking at 550 rpm in
an Eppendorf thermomixer, followed by an increase of the temperature to 37°C h at
constant shaking speed. The incubation was continued for 20 h. Samples were then
analyzed by flow cytometry.
Flow cytometry
Analysis of the samples was carried out on a FACSVantage flow cytometer (BD
Biosciences) at room temperature, measurement time was 30 sec per sample.
Authors' contributions
LT participated in the design of the study, carried out the measurements and drafted
the manuscript. ANS participated in the analysis of the data. EM prepared the
recombinant protein. KK and ST were also involved in protein expression and
purification. HS participated in the design and coordination of the study. GB
conceived of the study and helped to draft the manuscript. All authors read and
approved the final manuscript.
- 7 -
Acknowledgements
This work was kindly supported by grant No. 0312711A rom the BMBF
(Bundesministerium für Bildung und Forschung) in the context of the German
National TSE Research Platform. The authors gratefully acknowledge the help of the
TSE Research Platform, Munich, and the BFAV Riems, Germany, with respect to the
kind gift of biological material in the context of this study.
References
1 Prusiner SB: Novel proteinaceous infectious particles cause scrapie.
Science 1982, 216: 136-144
2 Kretzschmar HA, Prusiner SB, Stowring LE, DeArmond SJ: Scrapie prion
proteins are synthesized in neurons. Am J Pathol. 1986, 122: 1-5
3 Cashman NR, Loertscher R, Nalbantoglu J, Shaw I, Kascsak RJ, Bolton DC,
Bendheim PE: Cellular isoform of the scrapie agent protein participates in
lymphocyte activation. Cell. 1990, 61: 185-192
4 Manson J, West JD, Thomson V, McBride P, Kaufman MH, Hope J: The
prion protein gene: a role in mouse embryogenesis? Development 1992,
115: 117-122
5 Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I,
Huang Z, Fletterick RJ, Cohen FE, Prusiner SB: Conversion of alpha-helices
into beta-sheets features in the formation of the scrapie prion proteins.
Proc Natl Acad Sci U S A. 1993, 90: 10962-10966
6 Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS:
Secondary structure analysis of the scrapie-associated protein PrP 27-30
in water by infrared spectroscopy. Biochemistry 1991, 30: 7672-7680
- 8 -
7 Prusiner SB, Groth DF, Bolton DC, Kent SB, Hood LE: Purification and
structural studies of a major scrapie prion protein. Cell 1984, 38: 127-134
8 Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth
DF, Glenner GG: Scrapie prions aggregate to form amyloid-like
birefringent rods. Cell 1983, 35: 349-358
9 Prusiner SB: Prions. Proc Natl Acad Sci U S A. 1998, 95: 13363-13383
10 Kretzschmar HA, Ironside JW, DeArmond SJ, Tateishi J: Diagnostic criteria
for sporadic Creutzfeldt-Jakob disease. Arch Neurol. 1996, 53: 913-920
11 Brown P, Cervenakova L, Diringer H: Blood infectivity and the prospects
for a diagnostic screening test in Creutzfeldt-Jakob disease. J Lab Clin
Med. 2001, 137: 5-13
12 Proske D, Gilch S, Wopfner F, Schätzl HM, Winnacker EL, Famulok M:
Prion-protein-specific aptamer reduces PrPSc formation. Chembiochem.
2002, 3: 717-725
13 Gilch S, Wopfner F, Renner-Muller I, Kremmer E, Bauer C, Wolf E, Brem G,
Groschup MH, Schätzl HM: Polyclonal anti-PrP auto-antibodies induced
with dimeric PrP interfere efficiently with PrPSc propagation in prion-
infected cells. J Biol Chem. 2003, 278: 18524-18531
14 Post K, Pitschke M, Schafer O, Wille H, Appel TR, Kirsch D, Mehlhorn I,
Serban H, Prusiner SB, Riesner D: Rapid acquisition of beta-sheet structure
in the prion protein prior to multimer formation. Biol Chem. 1998, 379:
1307-1317
15 Pitschke M, Prior R, Haupt M, Riesner D: Detection of single amyloid beta-
protein aggregates in the cerebrospinal fluid of Alzheimer's patients by
fluorescence correlation spectroscopy. Nat Med. 1998, 4: 832-834
- 9 -
16 Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will
RG: Possible transmission of variant Creutzfeldt-Jakob disease by blood
transfusion. Lancet 2004, 363: 417-421
17 Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW: Preclinical vCJD
after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet
2004, 364: 527-529
18 Hunter N, Foster J, Chong A, McCutcheon S, Parnham D, Eaton S,
MacKenzie C, Houston F: Transmission of prion diseases by blood
transfusion. J Gen Virol. 2002, 83: 2897-2905
Figures
Figure 1 - Inhibition of PrP aggregation in serum
FITC-labeled recombinant bovine prion protein (concentration 10 nM) was incubated
at 37°C for 20 h with continuous shaking, either in 150 µl PBS (left panel) or in the
same volume of serum (right panel), followed by flow cytometry. The measurements
are depicted in a Fluorescence 1 (FL1-H) vs. Fluorescence 2 (FL2-H) dot-plot. The
number of counts in the area containing specific signals (R2) is given in the figures.
Aggregate formation in serum is strongly inhibited.
Figure 2 - Seed-dependent PrP aggregate formation in plasma
FITC-labeled recombinant prion protein (5 nM) was incubated in plasma as described
in the methods section for 20 h either in the absence (panel A) or presence (panel B)
of 10-8 nM PrP aggregates. Panel C: quantification of measurements shown in A and
B, and of measurements (not shown) with different seed concentrations. The
measurements are depicted in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-
- 10 -
plot. Aggregate formation (signal in region R1) was strongly enhanced by all seed
concentrations tested, from 5 nM to 10-8 nM.
Figure 3 - Analysis of serum from BSE-positive cattle
FITC-labeled recombinant prion protein (10 nM) was incubated in 150 µl of the
serum samples as described in the methods section and analyzed by flow cytometry.
The measurements are shown in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dotplot.
All six BSE-samples (A-F) can be differentiated from the controls (G-J) by a
population of events in region R3 (green dots). Panel K: Quantification of
measurements shown in panels A-J.
PBS
R2: 8436
Serum
R2: 162
Figure 1
A B
0
500
1000
1500
2000
2500
3000
3500
0.E+00
0.E+00
5.E+00
1.E+00
1.E-01
1.E-02
1.E-03
1.E-04
1.E-05
1.E-06
1.E-07
1.E-08
Fibril Concentration [nM]
Events in R1
C
Figure 2
K
0
100
200
300
400
500
600
700
800
A B C D E F G H I J
Serum sample
Events in R3
Figure 3
http://www.biomedcentral.com/content/pdf/1472-6750-5-26.pdf
TSS
#################### https://lists.aegee.org/bse-l.html ####################
From: TSS ()
Subject: Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy
Date: October 9, 2005 at 9:15 am PST
BMC Biotechnology
This Provisional PDF corresponds to the article as it appeared upon acceptance. The fully-formatted
PDF version will become available shortly after the date of publication, from the URL listed below.
Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood
from cattle affected with bovine spongiform encephalopathy
BMC Biotechnology 2005, 5:26 doi:10.1186/1472-6750-5-26
Lothar Trieschmann ([email protected].)
Alexander Navarrete Santos ([email protected].)
Katja Kaschig ([email protected].)
Sandra Torkler ([email protected].)
Elke Maas ([email protected])
Hermann Schatzl ([email protected])
Gerald Bohm ([email protected].)
ISSN 1472-6750
Article type Research article
Submission date 3 May 2005
Acceptance date 4 Oct 2005
Publication date 4 Oct 2005
Article URL http://www.biomedcentral.com/content/5/1/26
Like all articles in BMC journals, this peer-reviewed article was published immediately upon acceptance. It
can be downloaded, printed and distributed freely for any purposes (see copyright notice below).
Articles in BMC journals are listed in PubMed and archived at PubMed Central.
For information about publishing your research in BMC journals or any BioMed Central journal, go to
http://www.biomedcentral.com/info/authors/
© 2005 Trieschmann et al., licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
- 1 -
Ultra-sensitive detection of prion protein fibrils by
flow cytometry in blood from cattle affected with
bovine spongiform encephalopathy
Lothar Trieschmann1, Alexander Navarrete Santos1, Katja Kaschig1, Sandra Torkler1,
Elke Maas2, Hermann Schätzl2, Gerald Böhm1§
1ACGT ProGenomics AG, Weinbergweg 22, D-06120 Halle (Saale), Germany
2Institute of Virology, Technical University of Munich, Biedersteinerstrasse 29, D-
80802 Munich, Germany
§Corresponding author
Email addresses:
LT: [email protected].
ANS: [email protected].
KK: [email protected].
ST: [email protected].
HS: [email protected]
EM: [email protected]
GB: [email protected].
- 2 -
Abstract
Background
The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in
humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the
post mortem detection of the pathological form of the prion protein (PrPSc) in brain
tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even
at presymptomatic stages of the disease, albeit at concentrations well below the
detection limits of currently available analytical methods.
Results
We developed a highly sensitive method for detecting prion protein aggregates that
takes advantage of kinetic differences between seeded and unseeded polymerization
of prion protein monomers. Detection of the aggregates was carried out by flow
cytometry. In the presence of prion seeds, the association of labelled recombinant PrP
monomers in plasma and serum proceeds much more efficiently than in the absence
of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down
to a concentration of approximately 10-8 nM [0.24 fg/ml]. A specific signal was
detected in six out of six available serum samples from BSE-positive cattle.
Conclusions
We have developed a method based on seed-dependent PrP fibril formation that
shows promising results in differentiating a small number of BSE-positive serum
samples from healthy controls. This method may provide the basis for an ante mortem
diagnostic test for prion diseases.
- 3 -
Background
A group of fatal transmissible neurodegenerative diseases, including Creutzfeld-Jakob
disease (CJD), bovine spongiform encephalopathy (BSE), chronic wasting disease
(CWD) and scrapie, is caused by an unusual infectious agent that has been termed
prion [1]. Prions consist of an aberrant isoform (PrPSc) of the normal cellular prion
protein (PrPC). PrPC is a cell surface glycoprotein expressed in neurons [2] and other
cell types [3, 4]. The precise physiological function of the cellular prion protein is not
known yet. PrPSc differs from PrPC in its higher content of -sheet structure [5, 6], its
partial resistance to protease digestion [7], and its tendency to form large aggregates
[8]. PrPSc propagates by converting the cellular prion protein to the PrPSc
conformation [9]. PrPSc aggregates accumulate predominantly in the central nervous
system (CNS), and definitive diagnosis of prion diseases currently relies on the post
mortem detection of PrPSc in CNS tissue by immunohistochemistry, Western blotting,
or ELISA [10]. Transmission studies indicate that prions may also be present in
blood, potentially allowing for ante mortem diagnosis, but the sensitivity of the
currently available analytical methods is insufficient for the detection of the extremely
low prion titers that can be expected in body fluids [11].
Here, we report the development of a method based on kinetic differences between
seeded and unseeded aggregation of prion protein that allows the detection of PrP
aggregates in blood down to attomolar concentrations by flow cytometry.
Results and discussion
Detection of synthetic prion protein aggregates in serum or plasma
Kinetic differences between seeded and spontaneous polymerization of peptide
monomers can be used for the detection of amyloid -protein aggregates in the
- 4 -
cerebrospinal fluid of Alzheimer's disease patients [15]. Here, we extend the principle
of seeded polymerization to the detection of prion protein aggregates.
While trying to establish conditions for the labeling of synthetic prion protein
aggregates with a fluorescently labeled prion protein probe, we observed that the
formation of prion protein aggregates proceeds much less efficiently in serum or
plasma (not shown) than in PBS (Fig. 1). This inhibition is probably caused by
interactions of the prion protein probe with serum proteins.
Next, we found that the addition of preformed prion protein aggregates to plasma can
partially overcome this inhibition (Fig. 2). The preformed aggregates presumably
function as seeds that facilitate the formation of new aggregates in the inhibitory
environment of plasma. The seeds stimulated the formation of prion protein
aggregates at all concentrations tested, from 5 nM [120 ng/ml] to 10-8 nM [0.24 fg/ml]
(Fig 2C). The average ratio of event counts in seeded samples to those in samples
without seeds was 6.4. The number of events, however, was not proportional to the
seed concentration, but remained relatively constant over the whole concentration
range. Thus, the seed-dependent formation of prion protein aggregates can be used to
detect extremely low amounts (down to the attomolar range) of spiked prion protein
aggregates in blood.
Analysis of serum from clinical-stage, BSE-positive cattle
Studies demonstrating the transmission of prion diseases by blood transfusion suggest
that prions are present in the blood of afflicted animals and people, even at presymptomatic
stages of the disease [16, 17, 18]. We used the method of seeddependent
fibril formation to analyze serum from six confirmed cases of clinicalstage,
BSE-positive cattle and four controls. Based on the spiking experiments
described above, our hypothesis was that any PrPSc aggregates present in serum may
- 5 -
act as seeds for the formation of easily detectable amounts of labeled PrP aggregates,
whereas in the absence of seeds the formation of PrP aggregates would be inhibited.
The serum samples from BSE-positive cattle and controls from healthy cattle were
incubated with 10 nM of a FITC-labeled bovine PrP probe at 37°C for 20 h with
continuous shaking, followed by analysis in a flow cytometer. All six BSE-samples
could be clearly distinguished by a population of events that was absent in the
controls (Fig. 3A-J, green dots in region R3; quantification in fig. 3K).
Conclusions
We have developed a method based on seed-dependent PrP fibril formation that
shows promising results in differentiating a small number of BSE-positive serum
samples from healthy controls. More samples need to be tested in order to validate its
potential as an ante mortem diagnostic test for BSE and other prion diseases.
Methods
Biological fluids
Serum samples from six confirmed cases of BSE in cattle and four control animals
were obtained from BFAV, Insel Riems, Germany. Control plasma was obtained from
a blood bank.
Labeling of prion protein
Recombinant full-length bovine PrP was produced as described previously [12, 13].
The purified protein was labeled with a FITC-labeling kit (Roche) according to the
manufacturer's instructions.
- 6 -
Preparation of fibrils from recombinant prion protein
25 µM of unlabeled bovine prion protein in PBS containing 0.2 % SDS was incubated
for 10 min at room temperature, followed by a twentyfold dilution with PBS. For
fibril formation, the diluted reaction mixture was incubated for 48 h at room
temperature [14].
PrP fibril formation in serum or plasma
Recombinant FITC-labeled bovine prion protein was incubated in 150 µl serum or
plasma at a concentration of 5 or 10 nM for 5-10 min. at 20°C, shaking at 550 rpm in
an Eppendorf thermomixer, followed by an increase of the temperature to 37°C h at
constant shaking speed. The incubation was continued for 20 h. Samples were then
analyzed by flow cytometry.
Flow cytometry
Analysis of the samples was carried out on a FACSVantage flow cytometer (BD
Biosciences) at room temperature, measurement time was 30 sec per sample.
Authors' contributions
LT participated in the design of the study, carried out the measurements and drafted
the manuscript. ANS participated in the analysis of the data. EM prepared the
recombinant protein. KK and ST were also involved in protein expression and
purification. HS participated in the design and coordination of the study. GB
conceived of the study and helped to draft the manuscript. All authors read and
approved the final manuscript.
- 7 -
Acknowledgements
This work was kindly supported by grant No. 0312711A rom the BMBF
(Bundesministerium für Bildung und Forschung) in the context of the German
National TSE Research Platform. The authors gratefully acknowledge the help of the
TSE Research Platform, Munich, and the BFAV Riems, Germany, with respect to the
kind gift of biological material in the context of this study.
References
1 Prusiner SB: Novel proteinaceous infectious particles cause scrapie.
Science 1982, 216: 136-144
2 Kretzschmar HA, Prusiner SB, Stowring LE, DeArmond SJ: Scrapie prion
proteins are synthesized in neurons. Am J Pathol. 1986, 122: 1-5
3 Cashman NR, Loertscher R, Nalbantoglu J, Shaw I, Kascsak RJ, Bolton DC,
Bendheim PE: Cellular isoform of the scrapie agent protein participates in
lymphocyte activation. Cell. 1990, 61: 185-192
4 Manson J, West JD, Thomson V, McBride P, Kaufman MH, Hope J: The
prion protein gene: a role in mouse embryogenesis? Development 1992,
115: 117-122
5 Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I,
Huang Z, Fletterick RJ, Cohen FE, Prusiner SB: Conversion of alpha-helices
into beta-sheets features in the formation of the scrapie prion proteins.
Proc Natl Acad Sci U S A. 1993, 90: 10962-10966
6 Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS:
Secondary structure analysis of the scrapie-associated protein PrP 27-30
in water by infrared spectroscopy. Biochemistry 1991, 30: 7672-7680
- 8 -
7 Prusiner SB, Groth DF, Bolton DC, Kent SB, Hood LE: Purification and
structural studies of a major scrapie prion protein. Cell 1984, 38: 127-134
8 Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth
DF, Glenner GG: Scrapie prions aggregate to form amyloid-like
birefringent rods. Cell 1983, 35: 349-358
9 Prusiner SB: Prions. Proc Natl Acad Sci U S A. 1998, 95: 13363-13383
10 Kretzschmar HA, Ironside JW, DeArmond SJ, Tateishi J: Diagnostic criteria
for sporadic Creutzfeldt-Jakob disease. Arch Neurol. 1996, 53: 913-920
11 Brown P, Cervenakova L, Diringer H: Blood infectivity and the prospects
for a diagnostic screening test in Creutzfeldt-Jakob disease. J Lab Clin
Med. 2001, 137: 5-13
12 Proske D, Gilch S, Wopfner F, Schätzl HM, Winnacker EL, Famulok M:
Prion-protein-specific aptamer reduces PrPSc formation. Chembiochem.
2002, 3: 717-725
13 Gilch S, Wopfner F, Renner-Muller I, Kremmer E, Bauer C, Wolf E, Brem G,
Groschup MH, Schätzl HM: Polyclonal anti-PrP auto-antibodies induced
with dimeric PrP interfere efficiently with PrPSc propagation in prion-
infected cells. J Biol Chem. 2003, 278: 18524-18531
14 Post K, Pitschke M, Schafer O, Wille H, Appel TR, Kirsch D, Mehlhorn I,
Serban H, Prusiner SB, Riesner D: Rapid acquisition of beta-sheet structure
in the prion protein prior to multimer formation. Biol Chem. 1998, 379:
1307-1317
15 Pitschke M, Prior R, Haupt M, Riesner D: Detection of single amyloid beta-
protein aggregates in the cerebrospinal fluid of Alzheimer's patients by
fluorescence correlation spectroscopy. Nat Med. 1998, 4: 832-834
- 9 -
16 Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will
RG: Possible transmission of variant Creutzfeldt-Jakob disease by blood
transfusion. Lancet 2004, 363: 417-421
17 Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW: Preclinical vCJD
after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet
2004, 364: 527-529
18 Hunter N, Foster J, Chong A, McCutcheon S, Parnham D, Eaton S,
MacKenzie C, Houston F: Transmission of prion diseases by blood
transfusion. J Gen Virol. 2002, 83: 2897-2905
Figures
Figure 1 - Inhibition of PrP aggregation in serum
FITC-labeled recombinant bovine prion protein (concentration 10 nM) was incubated
at 37°C for 20 h with continuous shaking, either in 150 µl PBS (left panel) or in the
same volume of serum (right panel), followed by flow cytometry. The measurements
are depicted in a Fluorescence 1 (FL1-H) vs. Fluorescence 2 (FL2-H) dot-plot. The
number of counts in the area containing specific signals (R2) is given in the figures.
Aggregate formation in serum is strongly inhibited.
Figure 2 - Seed-dependent PrP aggregate formation in plasma
FITC-labeled recombinant prion protein (5 nM) was incubated in plasma as described
in the methods section for 20 h either in the absence (panel A) or presence (panel B)
of 10-8 nM PrP aggregates. Panel C: quantification of measurements shown in A and
B, and of measurements (not shown) with different seed concentrations. The
measurements are depicted in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dot-
- 10 -
plot. Aggregate formation (signal in region R1) was strongly enhanced by all seed
concentrations tested, from 5 nM to 10-8 nM.
Figure 3 - Analysis of serum from BSE-positive cattle
FITC-labeled recombinant prion protein (10 nM) was incubated in 150 µl of the
serum samples as described in the methods section and analyzed by flow cytometry.
The measurements are shown in a Fluorescence 1 (FL1-H) vs. Side-Scatter (SSC) dotplot.
All six BSE-samples (A-F) can be differentiated from the controls (G-J) by a
population of events in region R3 (green dots). Panel K: Quantification of
measurements shown in panels A-J.
PBS
R2: 8436
Serum
R2: 162
Figure 1
A B
0
500
1000
1500
2000
2500
3000
3500
0.E+00
0.E+00
5.E+00
1.E+00
1.E-01
1.E-02
1.E-03
1.E-04
1.E-05
1.E-06
1.E-07
1.E-08
Fibril Concentration [nM]
Events in R1
C
Figure 2
K
0
100
200
300
400
500
600
700
800
A B C D E F G H I J
Serum sample
Events in R3
Figure 3
http://www.biomedcentral.com/content/pdf/1472-6750-5-26.pdf
TSS
#################### https://lists.aegee.org/bse-l.html ####################
