Use of PMCA for Biochemical
Diagnosis of Prion Diseases
Claudio Soto, PhD
Dept of Neurology, University of Texas Medical Branch
and Amprion Inc.
Importance of Prion Diagnosis
Food Blood banks Plasma
industry products
Disease Clinical trials
diagnosis
Brain surgery Organ
Drugs from transplant
Soto (2005) Nature Rev Microbiol. 2:809-819 human origin
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)
PrPSc amplification during disease propagation
PrPSc PrPC
Slow process
Disease
Infection Incubation Time
60 - 120 d
Clinical
symptoms
3-5y
7 - 40 y
Protein Misfolding Cyclic Amplification (PMCA)
PrPSc
+
Incubation Sonication Incubation Sonication
Growing Multiplication Growing
of units Multiplication
of units of units
of units
PrPC
Soto et al. (2002) Trends Neurosci. 25:390-394
PMCA: Proof of concept
source of PrPSc source of PrPc
Sick hamster (263k) Healthy hamster
PMCA - +
Brain homogenates
Amplification
(Incubation + sonication)
PK digestion
PrPres fragment
PK + +
WB analysis
Saborio, Permanne and Soto (2001) Nature 411:810-813
Automated PMCA
Amplified Non
Amplified
Ultrasensitive detection of PrPSc by serial PMCA
NBH
-PK
Non
amplified
Non
amplified
Dilution
NBH
Dilution -PK
1st PMCA
144 PMCA
(96 cycles)
cycles
NBH
Dilution -PK
Amplification factor ~ 6600 fold
2nd PMCA 1:10
(118 cycles)
Castilla, Saa and Soto (2005) Amplification
Nature Medicine 11:982-985 factor ~10 million folds
What is the minimum quantity detected?
Scrapie LD50 spiked
100000 10000 1000 100 10 1 0 NBH
-PK
Non amplified
4th PMCA
(144 cycles)
1st PMCA
(144 cycles)
Scrapie LD50 spiked
2nd PMCA
(144 cycles)
7th PMCA
3rd PMCA
(144 cycles)
Our mathematical estimation is that 0.001 LD50 contains
4th PMCA
(144 cycles) around 20-50 molecules of PrP monomer
Saa, Castilla, and Soto (2006)
J. Biol. Chem (In press)
Table 1: of PrP the detection by different
SensitivityComparison of Sc sensitivity of several methods to detect PrP methods Sc
Assay Maximum Minimum PrP Minimum Increase in
dilution quantity number of sensitivityd
detected a detected b equivalent PrP
Table 1: Comparison of the sensitivity of several methodsmoleculesc Sc
to detect PrP
Standard western blot 3.0 x 103 4.0 ng 8.0 x 1010 1
Assay Maximum Minimum PrP Minimum Increase in
dilution quantity number of sensitivityd
ELISA 2.5 x 104
detecteda 0.5 ng
detectedb 1.0 x 1010
equivalent PrP 8
moleculesc
Phosphotunstic acid
Standard western blot 1.5 x 105
3.0 x 103 80 ng
4.0 pg 1.6 x 109
8.0 x 1010 50
1
precipitation
Conformation dependent
ELISA 2.0 x 105
2.5 4 150 ng
0.5 pg 3.0 x 109
1.0 x 1010 27
8
immunoassaye
Animal bioassayacid 9
1.0 x 105 12 fg 5
2.4 x 109 330,000
Phosphotunstic 1.5 80 pg 1.6 50
precipitation
Single PMCAf dependent
Conformation 7.0 x 105
2.0 6
150 pg
1.6 pg 7
3.2 x 109
3.0 2,500
27
1 round PMCA
immunoassaye
Animal PMCAf
Double bioassay 2.0 x 1010
1.0 x 109 12 fg
0.6 fg 4
1.2 x 105
2.4 6,500,000
330,000
2 rounds PMCA
Single PMCAf
a The maximum 7.0 x 106
dilution detected 1.0 x 10 12 to the last dilution of 263K scrapie brain in which PrP is detectable.
corresponds 1.6 pg 3.2 x 107 2,500
Sc
7 rounds PMCA 1.3 ag 26 3,300,000,000
b The minimum quantity of PrPSc detected in a brain sample volume of 20 µl.
c The number of PrP molecules was estimated by comparison with recombinant PrP4 and Is expressed as equivalent
Double PMCAf 2.0 x 1010 0.6 fg 1.2 x 10 6,500,000
molecules to highlight that this is a mathematically estimated quantity.
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 was taken from the literature, whereas all the others were
a The maximum dilution detected corresponds to the last dilution of 263K scrapie brain in which PrPSc is detectable.
experimentally calculated.
b The minimum quantity of PrPSc detected in a brain sample volume of 20 µl.
f The data for single and double PMCA
Saa, Castilla, and Soto (2006) PMCA. correspond to the average obtained in three different experiments equivalent
c The number of PrP molecules was estimated by comparison with recombinant PrP and Is expressed as
cycles in both first and second
using 100
Chem (In press)
J. Biol.molecules to highlight that this is a mathematically estimated quantity.
d The increase of sensitivity is expressed in relation to the standard western blot assay using 3F4 antibody.
e
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
Our sensitivity
Single PMCA: 65 pg/ml = 1.3 x 109 molecules/ml
Double PMCA: 0.02 pg/ml = 4 x 105 molecules/ml
7th rounds PMCA: 0.015 fg/ml = 500 molecules/ml
Can we detect PrPSc in blood?
NBH
S1 S2 C1 C2 S3 S4 S5 S6 C3 C4 C5 S7 S8 S9 S10 S11 S12 S13 S14 S15 C6 C7 S16 S17 C8 S18 C9 C10 C11 C12 -PK
1st 36.2
29.9
36.2
2nd
29.9
36.2
3rd
29.9
36.2
4th 29.9
36.2
5th 29.9
36.2
6th
29.9
Castilla, Saa and Soto (2005) Nature Medicine 11:982-985
Pre-symptomatic detection of PrPSc in blood
Infection Blood taken during incubation period Clinical disease
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
Time, days Controls Infected NBH Sensitivity/ NBH NBHNBH
NBH
NBH NBH
I1 C1 I1 I3 C3
I2 I1 -PK -PK C3 C2 C4 C4 I1C2 C3 I5 -PK I6 C2 I2 C3
I5 C1 C5C2 C4 C6 I3 C4 I4 C8
C1 I4 C5 C3
I2 C1 C7 I7 C5
C1 C5 C2 I1 I2C1I3I3I4C3Total/positives I1 specificity
I2
I2 C2 I3 I4I3 I5I4 I6C4C1I5C2 Total/positives
I8 C4
I5 I6 I7 C3 I9 I10 -PK I8 C4 -PK C10 I10 -PK -PK
I5
I3 C9 I4 C5 I5
I9
I1 I2
36.2 36.2
14 5/0 5/0 0% / 100% 36.2
36.2
36.2
36.2 36.2
29.9
20 4/0 29.9 6/3 29.9
50% / 100%
29.9 29.9 29.9
29.9
40 5/0 10/6 60% / 100%
60 4/0 5/2 40% / 100%
70 5/0 5/1 20% / 100%
Saa, Castilla and Soto
80 5/0 5/0 0% / 100% (2006) Science 313:92-94
Symptomatic 10/0 10/8 80% / 100%
Pre-symptomatic detection of PrPSc in blood
Peripheral prion replication? Leakage from the brain?
% of PrPSc positive animals
100
50
0
0 25 50 75 100
Time, days
Saa, Castilla and Soto Symptomatic phase
(2006) Science 313:92-94
Application of PMCA to different samples
- + - + - +
sCJD sCJD vCJD
type 1 type 2
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
Rodrigo Morales Karim Abid, PhD
June Yowtak Lisbell Estrada
Joaquín Becky Daniels
Paula Saá
Castilla, PhD
Jorge de Castro Claudio Soto, PhD
Former lab members Collaborators
Gabriela Saborio, MD Case Western Reserve University
Pierluigi Gambetti
Celine Adessi, PhD
Kinsey Maundrell, PhD CJD Unit, Edinburgh, UK
Bruno Permanne, PhD Robert Will
Youcef Fezoui, PhD James Ironside
Raphaele Buser, PhD
Milene Russelaskis, PhD Istituto Carlo Besta, Italy
Fabrizio Tagliavini
Claudio Hetz, PhD
Sergio Benavent US Department of Agriculture
Laurence Anderes Juergen Richt
Marie-Jose Frossard
Santiago Fraga Istituto Superiore di Sanita, Italy
Maurizio Pocchiari
Elizabeth Vial
Sergio Peano, MD University of Kentucky
Thomas Ruckle, PhD Glenn Telling
University of Edinburgh, UK
Jean Manson
University of Zurich, Switzerland
Adriano Aguzzi
Mathias Heikenwalder