Immuno-Real Time-PCR as a sensitive diagnostic tool: … · Center of Research on Prion Protein,...
Transcript of Immuno-Real Time-PCR as a sensitive diagnostic tool: … · Center of Research on Prion Protein,...
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ImmunoImmuno--Real TimeReal Time--PCR as a sensitive PCR as a sensitive diagnostic tool: case of diagnostic tool: case of prionprion proteins.proteins.
Freising-Weihenstephan – 26th-30th March 2007
qPCR2007 - 3rd International qPCR Symposium
Virginie Ruelle and Benaissa ElMoualij.
Center of Research on Prion Protein, University of Liège, Liège, Belgium
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A. Prion diseases : Types
⇒ Transmissible neurodegenerative disorders whichaffect the Central Nervous System (CNS)
1. Introduction
qPCR2007 - 3rd International qPCR Symposium
→ Bovine Spongiform Encephalopathy (BSE)
→ Scrapie
→ Creutzfeldt-Jakob
No-infectious Infectious
CJD Variant CJD(vCJD)
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B. Prion diseases : Symptoms
1. Introduction
→ Dementia
Me ? Me ? No, INo, I’’m a m a rabbit !rabbit !
Are you Are you afraid of afraid of BSE ?BSE ?
qPCR2007 - 3rd International qPCR Symposium
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→ Locomotive disorders→ Dementia
qPCR2007 - 3rd International qPCR Symposium
B. Prion diseases : Symptoms
1. Introduction
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→ Spongiform pathology of brain VacuoleVacuole
AggregateAggregate
⇒ Fatal
→ Locomotive disorders→ Dementia
qPCR2007 - 3rd International qPCR Symposium
B. Prion diseases : Symptoms
1. Introduction
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C. Prion diseases : Infectious agent
1. Introduction
→ Abnormal protein : Abnormal protein : ““PrPPrPscsc” ou “PrPPrPresres”
. Scrapie Prion
. Pathogenic
. Relatively no-Soluble
. Proteinase K resistant
→ Normal protein : Normal protein : ““PrPPrPcc””
. Cellular prionCellular prion
. No-pathogenic
. Soluble⇒ Sequence =⇒ Folding ≠
qPCR2007 - 3rd International qPCR Symposium
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PrPPrPcc
qPCR2007 - 3rd International qPCR Symposium
1. Introduction
C. Prion diseases : Infectious agent
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PrPPrPscsc
qPCR2007 - 3rd International qPCR Symposium
PrPPrPcc
1. Introduction
C. Prion diseases : Infectious agent
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9qPCR2007 - 3rd International qPCR Symposium
PrPPrPcc
PrPPrPscsc
1. Introduction
C. Prion diseases : Infectious agent
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PrPPrPcc
PrPPrPscsc
1. Introduction
C. Prion diseases : Infectious agent
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PrPPrPcc
PrPPrPscsc
1. Introduction
C. Prion diseases : Infectious agent
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PrPPrPcc
PrPPrPscsc
1. Introduction
C. Prion diseases : Infectious agent
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PrPPrPcc
PrPPrPscsc
1. Introduction
AggregateAggregate
C. Prion diseases : Infectious agent
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1. Introduction
D. Prion diseases : vCJD Contamination
Ludlam CA et al. British Journal of Haematology, 132, 13–24.
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1. Introduction
D. Prion diseases : vCJD Contamination
CowCowBSEBSE
HumanHumanvCJDvCJD
ConfirmedConfirmed casescases
188.417188.417FromFrom 19871987
198198FromFrom 19951995
ContaminatedFood
Ludlam CA et al. British Journal of Haematology, 132, 13–24.
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1. Introduction
D. Prion diseases : vCJD Contamination
CowCowBSEBSE
HumanHumanvCJDvCJD
ConfirmedConfirmed casescases
188.417188.417FromFrom 19871987
198198FromFrom 19951995
ContaminatedFood
Diagnostic
6666BloodBlood recipientsrecipients
44FromFrom 20042004
ContaminatedBlood
3131BloodBlood donorsdonors
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1. Introduction
Pré-clinicalphase (LONG)
Clinical Phase
(SHORT)
InoculationIngestion of BSE
contaminated food
Firstclinical signs
Death
Disease blockingDisease blocking
Bruce ME et al. Immunol Today. 2000;21(9):442-6.
New sensitive test to detect prion:
Say Say MeuhMeuh
No No MeuhMeuh !!!!
D. Prion diseases : vCJD Contamination
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→ Living :
→ Post-mortem : BRAIN- Histological coupe
- Molecular diagnostic
Symptoms
qPCR2007 - 3rd International qPCR Symposium
1. Introduction
E. Prion diseases : Diagnostic
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Prionics CheckWestern
Western BlotWestern Blot
Institut PourquierSpeed’it BSE
Prionics CheckLIA
Roboscreen BetaPrionBSE EIA test kit
Roche Applied SciencePrioScreen
ENFER TSE kitv.2.0 InPro CDI-5
BioRad TeSeE™Detection KitELISAELISA
Ceditect BSE test IDEXX Herdchek BSE Antigen Test Kit EIA
Prionics CheckPrioStrip
OtherOther
Rapid BSE testing kits
approved by the European Commission
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Classical assays on PrPsc Threshold of sensitivity
Western blot 1000-3000 IU/mL
ELISA ~ 1000 IU/mL
qPCR2007 - 3rd International qPCR Symposium
1. Introduction
E. Prion diseases : Molecular Diagnostic SENSITIVITY
Clinical phase :
> 100 IU/mL
Pré- Clinical phase :
< 10 IU/mLimmuno-PCR
Barletta JM et al. J. Virol. Methods. 2005;127:154-164.
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PCR
Plate
Antigen
Capture Antibody
DNADNA
Detectionantibody
Signal amplificationSignal amplification
2. Immuno-PCR
A. Principle :
qPCR2007 - 3rd International qPCR Symposium
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Plate
Antigen
Capture Antibody
BiotinylatedDetectionantibody
2. Immuno-PCR
A. Principle :
B
qPCR2007 - 3rd International qPCR Symposium
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Plate
Antigen
Capture Antibody
BiotinylatedDetectionantibody
2. Immuno-PCR
A. Principle :
B Strep
qPCR2007 - 3rd International qPCR Symposium
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Plate
Antigen
Capture Antibody
BiotinylatedDetectionantibody
2. Immuno-PCR
A. Principle :
B Strep
Chimeric DNA NatureMatrix
qPCR2007 - 3rd International qPCR Symposium
Patent: EP1232283,2002-08-21.i-Detect Company
www.i-detect.eu
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Synthetic
≠ Organisms
A B C
Chimeric DNA
2. Immuno-PCR
A. Principle : NatureMatrix
qPCR2007 - 3rd International qPCR Symposium
Patent: EP1232283,2002-08-21.i-Detect Company
www.i-detect.eu
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0 1 2 3 4 5 6 7
24
25
26
27
28
29
30
31 0
1
2
Recombinante Prion
ELISA10 ng/ml
iPCRq100 pg/ml
Detection limit
Log C
Cyc
le t
Optical D
ensity2. Immuno-PCR
B. Evaluation on human recombinant prion protein
immunoimmuno--PCRPCR
Detection limit ~ 100pg/mL
ELISAELISA
Detection limit ~ 10 ng/mL
Improvement 100 x
qPCR2007 - 3rd International qPCR Symposium
1E5Plate
PrPrec
4F7 B Strep
1E5Plate
PrPrec
4F7 B StrepStrep
No-pathogenic
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Pathogenic
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
Studied cases
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Pathogenic
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
Studied cases
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Pathogenic
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Sample preparation
HomogenizationPK digestionConcentration
Plate
Pathogenic
MiddleMiddle frontal gyrusfrontal gyrusDiagnostic surgical biopsies
Have been performed previously
DilutionDilution
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°1
ELISA100x
iPCRq5.000x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Improvement 50 x
1 10 100 1000
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25
26
27
28
29
30
31
32 0.0
0.5
1.0
1.5
2.0Prion in brainControl n°1
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)
ELISA/iqPCR comparison
Pathogenic
1E5Plate
PrPrec
4F7 B Strep
1E5Plate
PrPrec
4F7 B StrepStrep
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°1
ELISA100x
iPCRq5.000x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Improvement 50 x
ELISA/iqPCR comparison
Pathogenic
1 10 100 1000
24252627282930313233343536 0.0
0.5
1.0
1.5
2.0
Prion in brainControl n°1 to 7
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)
1E5Plate
PrPrec
4F7 B Strep
1E5Plate
PrPrec
4F7 B StrepStrep
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°1
ELISA100x
iPCRq5.000x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Improvement 50 x
ELISA/iqPCR comparison
Pathogenic
1E5Plate
PrPrec
4F7 B Strep
1E5Plate
PrPrec
4F7 B StrepStrep
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1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°1 to 7
Cyc
le th
resh
old
(Ct)
Optical D
ensity(450nm
)2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
Validation
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°3
ELISA10x
iPCRq1.000x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°2
ELISA100x
iPCRq5.000x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°4
ELISA5x
iPCRq500x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°5
ELISA50x
iPCRq5.000x
Dilution (1/x)
()
Optical D
ensity(450nm
)
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°6
ELISA10x
iPCRq1.000x
Dilution (1/x)
()
Optical D
ensity(450nm
)
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°7
ELISA50x
iPCRq1.000x
Dilution (1/x)
()
Optical D
ensity(450nm
)
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Improvement 20 to 100 x
100 x
100 x
20 x
100 x
50 x 100 x
Pathogenic
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
Validation
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Pathogenic
2520
10
375075
CJD Controlsrec 1 2 3 4 5 6 7 1 2 3 4 5 6 7
2520
10
375075
2520
10
375075
CJD Controlsrec 1 2 3 4 5 6 7 1 2 3 4 5 6 7
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
Validation
qPCR2007 - 3rd International qPCR Symposium Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
Pathogenic
2520
10
375075
CJD Controlsrec 1 2 3 4 5 6 7 1 2 3 4 5 6 7
2520
10
375075
2520
10
375075
CJD Controlsrec 1 2 3 4 5 6 7 1 2 3 4 5 6 7
1 10 100 1000
242526272829303132 0.0
0.5
1.0
1.5
2.0
Prion in brainCJD n°4
ELISA5x
iPCRq500x
Dilution (1/x)
Cyc
le th
resh
old
(Ct)
100 x
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2. Immuno-PCR
C. Application : diagnostic of Creutzfeldt-Jakob Disease
qPCR2007 - 3rd International qPCR Symposium
Pathogenic
. All methods shows similar sensitivity and specificity(100% detected)
Signal improvement compared to ELISA and Western-Blot
~ 100x on pathogenic protein (CJD)
. Comparison of brain samples7 patients with sporadic CJD / 7 controls
using : ELISA
Western-Blot
immuno-PCR
. immuno-PCR as a new useful toolfor ante-mortem PrPres detection
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2. Immuno-PCR
D. Protocol improvement vs reproducibility: Buffer/Antibodies/DNA/Plate
qPCR2007 - 3rd International qPCR Symposium
4F7 biotinylated antibodyRoboscreen
Plate
Strep
controls
Plate
4F7BStrep
Detection complexes
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40qPCR2007 - 3rd International qPCR Symposium
8 controls
Day n°11 at Ct=38
7 at Ct=40
Day n°2
Day n°3
1 at Ct=38,5
7 at Ct=40
1 > Ct=37
7 at Ct=40
Background
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41qPCR2007 - 3rd International qPCR Symposium
8 detection complexes
Day n°1
Day n°2
Day n°3
8 Ct in [25-26]
8 Ct in [25-26]
8 Ct in [25-26,2]
Reproducibility
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2. Immuno-PCR
E. Detection in blood:
qPCR2007 - 3rd International qPCR Symposium
EUROPE6e Framework Programme
2007-2010
EUDICAEUDICA
Jozef Stefan InstituteLjubljana
Plasma
lab
Jozef Stefan InstituteLjubljana
Plasma
lab
LANCASTER UNIVERSITY
CHULyon
National Institute of Psychiatry
and NeurologyBUDAPEST
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43
2. Immuno-PCR
qPCR2007 - 3rd International qPCR Symposium
EUROPE6e Framework Programme
2007-2010
EUDICAEUDICA
Jozef Stefan InstituteLjubljana
Plasma
lab
Jozef Stefan InstituteLjubljana
Plasma
lab
LANCASTER UNIVERSITY
CHULyon
National Institute of Psychiatry
and NeurologyBUDAPEST
Diagnostic kits
Creutzfeldt-JakobAlzheimerParkinson
E. Detection in blood:
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. Immuno-PCR as ultrasensitive toolSignal improvement compared to ELISA
- 100x on pathogenic protein (CJD)
. Immuno-PCR as detection toolcould be used to detect pathogenic and no-pathogenic
forms of Prion protein (and others)
3. Conclusions
qPCR2007 - 3rd International qPCR Symposium
. Immuno-PCR with low backgroundBuffers/Antibodies/DNA/plate improvement
- Background near of a Ct= 40
- Reproductibility intra – and inter-days
. immuno-PCR as a new usefulfor ante-mortem PrPres detection IN BLOOD
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AcknowledgementsAcknowledgements
qPCR2007 - 3rd International qPCR Symposium
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AcknowledgementsAcknowledgements
qPCR2007 - 3rd International qPCR Symposium
EUDICAEUDICA
Jozef Stefan InstituteLjubljana
Plasma
lab
Jozef Stefan InstituteLjubljana
Plasma
lab
LANCASTER UNIVERSITY
CHULyon
National Institute of Psychiatry
and NeurologyBUDAPEST
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ThankThank youyou for for youryour attentionattention
Contacts :
qPCR2007 - 3rd International qPCR Symposium