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


→ Bovine Spongiform Encephalopathy (BSE)

→ Scrapie

→ Creutzfeldt-Jakob

No-infectious Infectious

CJD Variant CJD(vCJD)

3

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 ?


4

→ Locomotive disorders→ Dementia



1. Introduction

5

→ Spongiform pathology of brain VacuoleVacuole

AggregateAggregate

⇒ Fatal

→ Locomotive disorders→ Dementia



1. Introduction

6

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 ≠


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PrPPrPcc


1. Introduction


8

PrPPrPscsc


PrPPrPcc

1. Introduction


9qPCR2007 - 3rd International qPCR Symposium

PrPPrPcc

PrPPrPscsc

1. Introduction



PrPPrPcc

PrPPrPscsc

1. Introduction



PrPPrPcc

PrPPrPscsc

1. Introduction



PrPPrPcc

PrPPrPscsc

1. Introduction



PrPPrPcc

PrPPrPscsc

1. Introduction

AggregateAggregate



1. Introduction

D. Prion diseases : vCJD Contamination

Ludlam CA et al. British Journal of Haematology, 132, 13–24.


1. Introduction


CowCowBSEBSE

HumanHumanvCJDvCJD

ConfirmedConfirmed casescases

188.417188.417FromFrom 19871987

198198FromFrom 19951995

ContaminatedFood

Ludlam CA et al. British Journal of Haematology, 132, 13–24.


1. Introduction


CowCowBSEBSE

HumanHumanvCJDvCJD

ConfirmedConfirmed casescases

188.417188.417FromFrom 19871987

198198FromFrom 19951995

ContaminatedFood

Diagnostic

6666BloodBlood recipientsrecipients

44FromFrom 20042004

ContaminatedBlood

3131BloodBlood donorsdonors


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 !!!!


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→ Living :

→ Post-mortem : BRAIN- Histological coupe

- Molecular diagnostic

Symptoms


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

21

Classical assays on PrPsc Threshold of sensitivity

Western blot 1000-3000 IU/mL

ELISA ~ 1000 IU/mL


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.

22

PCR

Plate

Antigen

Capture Antibody

DNADNA

Detectionantibody

Signal amplificationSignal amplification

2. Immuno-PCR

A. Principle :


23

Plate

Antigen

Capture Antibody

BiotinylatedDetectionantibody

2. Immuno-PCR

A. Principle :

B


24

Plate

Antigen

Capture Antibody


2. Immuno-PCR

A. Principle :

B Strep


25

Plate

Antigen

Capture Antibody


2. Immuno-PCR

A. Principle :

B Strep

Chimeric DNA NatureMatrix


Patent: EP1232283,2002-08-21.i-Detect Company

www.i-detect.eu

26

Synthetic

≠ Organisms

A B C

Chimeric DNA

2. Immuno-PCR

A. Principle : NatureMatrix


Patent: EP1232283,2002-08-21.i-Detect Company

www.i-detect.eu

27

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


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

29

2. Immuno-PCR


Studied cases


Pathogenic

30

2. Immuno-PCR


Studied cases


Pathogenic

31

2. Immuno-PCR



Sample preparation

HomogenizationPK digestionConcentration

Plate

Pathogenic

MiddleMiddle frontal gyrusfrontal gyrusDiagnostic surgical biopsies

Have been performed previously

DilutionDilution

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2. Immuno-PCR


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

)


Improvement 50 x

1 10 100 1000

24

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


1 10 100 1000

242526272829303132 0.0

0.5

1.0

1.5

2.0


ELISA100x

iPCRq5.000x

Dilution (1/x)

Cyc

le th

resh

old

(Ct)

Optical D

ensity(450nm

)


Improvement 50 x


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

34

2. Immuno-PCR


1 10 100 1000

242526272829303132 0.0

0.5

1.0

1.5

2.0


ELISA100x

iPCRq5.000x

Dilution (1/x)

Cyc

le th

resh

old

(Ct)

Optical D

ensity(450nm

)


Improvement 50 x


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


Validation

1 10 100 1000

242526272829303132 0.0

0.5

1.0

1.5

2.0


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


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


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


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


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


ELISA50x

iPCRq1.000x

Dilution (1/x)

()

Optical D

ensity(450nm

)


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


Validation


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

37

2. Immuno-PCR


Validation


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


ELISA5x

iPCRq500x

Dilution (1/x)

Cyc

le th

resh

old

(Ct)

100 x

38

2. Immuno-PCR



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


4F7 biotinylated antibodyRoboscreen

Plate

Strep

controls

Plate

4F7BStrep

Detection complexes


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


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:


EUROPE6e Framework Programme

2007-2010

EUDICAEUDICA

Jozef Stefan InstituteLjubljana

Plasma

lab


Plasma

lab

LANCASTER UNIVERSITY

CHULyon

National Institute of Psychiatry

and NeurologyBUDAPEST

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2. Immuno-PCR


EUROPE6e Framework Programme

2007-2010

EUDICAEUDICA


Plasma

lab


Plasma

lab


CHULyon



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


. 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


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AcknowledgementsAcknowledgements


EUDICAEUDICA


Plasma

lab


Plasma

lab


CHULyon



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ThankThank youyou for for youryour attentionattention

Contacts :

[email protected]


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Immuno-Real Time-PCR as a sensitive diagnostic tool: … · Center of Research on Prion Protein,...

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