Use of PMCA for Biochemical Diagnosis of Prion Diseases

1
Use of PMCA for Biochemical Diagnosis of Prion
Diseases
Claudio Soto, PhD
Dept of Neurology, University of Texas Medical
Branch and Amprion Inc.
2
Importance of Prion Diagnosis
Blood banks
Food industry
Plasma products
TSE diagnosis
Disease diagnosis
Clinical trials
Organ transplant
Brain surgery
Drugs from human origin
Soto (2005) Nature Rev Microbiol. 2809-819
3
The problem of Prion Diagnosis

• PrPsc Is the most specific marker for the
  disease. However, its levels in body fluids and
  tissues other than nervous system are too low to
  be detected
• Design a more sensitive test for PrPsc detection
• or
• Amplify the level of the marker (PrPSc)

4
PrPSc amplification during disease propagation
PrPC
PrPSc
Slow process
Disease
Incubation Time
Infection
60 - 120 d
Clinical symptoms
3 - 5 y
7 - 40 y
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Protein Misfolding Cyclic Amplification (PMCA)
PrPSc

PrPC
Soto et al. (2002) Trends Neurosci. 25390-394
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PMCA Proof of concept
source of PrPc
source of PrPSc
Healthy hamster
Sick hamster (263k)
PMCA -
Brain homogenates
Amplification (Incubation sonication)
PK digestion
PrPres fragment
PK
WB analysis
Saborio, Permanne and Soto (2001) Nature
411810-813
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Automated PMCA
Non Amplified
Amplified
control
One-by-one
96 format
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Ultrasensitive detection of PrPSc by serial PMCA
Castilla, Saa and Soto (2005) Nature Medicine
11982-985
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What is the minimum quantity detected?
Scrapie LD50 spiked
NBH -PK
100000
10000
1000
100
10
1
0
Saa, Castilla, and Soto (2006) J. Biol. Chem (In
press)
10
Sensitivity of PrPSc detection by different
methods
Saa, Castilla, and Soto (2006) J. Biol. Chem (In
press)
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Can we detect PrPSc in blood?
How much PrPSc is there in blood?
Symptomatic phase
Plasma 0.1 pg/ml 2 x 107 molecules Buffy
coat 1 pg/ml 2 x 108 molecules
Incubation period (pre-symptomatic phase)
Plasma 0.005-0.01 pg/ml 1-2 x 106
molecules Buffy coat 0.05-0.1 pg/ml 1-2 x 107
molecules
Taken from Brown et al (2001) J. Lab Clin.
Invest. 137 5-13
In other words sensitivity of PrPSc detection as
compared with standard western blot has to be
increase by 100,000 1,000,000 fold
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Can we detect PrPSc in blood?
Castilla, Saa and Soto (2005) Nature Medicine
11982-985
13
Pre-symptomatic detection of PrPSc in blood
Infection
Clinical disease
Blood taken during incubation period
14 days
20 days
40 days
60 days
70 days
80 days
Symptomatic
0/5
3/6
6/10
2/5
1/5
0/5
8/10
Saa, Castilla and Soto (2006) Science 31392-94
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Pre-symptomatic detection of PrPSc in blood
Symptomatic phase
Saa, Castilla and Soto (2006) Science 31392-94
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Application of PMCA to different samples
- - -

sCJD type 1
sCJD type 2
vCJD
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What is next?

• Adapt and optimize blood detection of PrPSc in
  relevant natural samples (cow, human, sheep,
  deer)
• Large scale study to evaluate the detection of
  PrPSc in blood of healthy donors in countries
  with high risk of vCJD (UK, France, etc)
• Study earliest time in which PrPSc can be
  detected in humans (primate model, familial
  cases) and in cattle (experimental infection
  model)
• Optimize the method for detection in other blood
  components (plasma, red cells) and other
  biological fluids (urine, CSF).
• Develop the technology into a high throughput and
  practical test

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Rodrigo Morales
Karim Abid, PhD
Lisbell Estrada
June Yowtak
Joaquín Castilla, PhD
Becky Daniels
Paula Saá
Claudio Soto, PhD
Jorge de Castro
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Collaborators
Former lab members
Case Western Reserve University Pierluigi
Gambetti CJD Unit, Edinburgh, UK Robert
Will James Ironside Istituto Carlo Besta,
Italy Fabrizio Tagliavini US Department of
Agriculture Juergen Richt Istituto Superiore di
Sanita, Italy Maurizio Pocchiari University of
Kentucky Glenn Telling University of Edinburgh,
UK Jean Manson University of Zurich,
Switzerland Adriano Aguzzi Mathias Heikenwalder
Gabriela Saborio, MD Celine Adessi, PhD Kinsey
Maundrell, PhD Bruno Permanne, PhD Youcef
Fezoui, PhD Raphaele Buser, PhD Milene
Russelaskis, PhD Claudio Hetz, PhD Sergio
Benavent Laurence Anderes Marie-Jose
Frossard Santiago Fraga Elizabeth Vial Sergio
Peano, MD Thomas Ruckle, PhD