SEARCH VEGSOURCE:

 

 

Follow Ups | Post Followup | Back to Discussion Board | VegSource
See spam or
inappropriate posts?
Please let us know.
  




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 (lothar.trieschmann@acgt-progenomics.de)

Alexander Navarrete Santos (alexander.navarrete.santos@acgt-progenomics.de)

Katja Kaschig (katja.kaschig@acgt-progenomics.de)

Sandra Torkler (sandra.torkler@acgt-progenomics.de)

Elke Maas (maas@lrz.tu-muenchen.de)

Hermann Schatzl (schaetzl@lrz.tum.de)

Gerald Bohm (gerald.boehm@acgt-progenomics.de)

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: lothar.trieschmann@acgt-progenomics.de

ANS: alexander.navarrete.santos@acgt-progenomics.de

KK: katja.kaschig@acgt-progenomics.de

ST: sandra.torkler@acgt-progenomics.de

HS: schaetzl@lrz.tum.de

EM: maas@lrz.tu-muenchen.de

GB: gerald.boehm@acgt-progenomics.de

- 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




Follow Ups:



Post a Followup

Name:
E-mail: (optional)
Subject:

Comments:

Optional Link URL:
Link Title:
Optional Image URL: