1HPerron TSEAC 09 2006

Detection of PrPres in plasma

H. Perron

Presented by P van Driessche

2HPerron TSEAC 09 2006

PrPres in plasma Chemical ligands and Immunoassay

COLLABORATIVE NETWORK

Laboratory for the Diagnosis of Human Prion

Diseases

Neurological Hospital, Bron/Lyon.

Dr Armand Perret-Liaudet

Dr Isabelle Quadrio

Séverine Ugnon-Café

ATU New Markers

« R&D neurological diseases »

BIOMERIEUX SA, Marcy/Lyon

AFSSA, Lyon

Dr Aly Moussa

Dr Thierry Baron

IBCP (CNRS & Claude Bernard Univ.) , Lyon

Dr Tony Coleman

Sébastien Cécillon

Dr Hervé Perron

Marilyne Dupin

Géraldine Ramage

Séverine Darneix

Isabelle Surault

3HPerron TSEAC 09 2006

Towards an Immunoassay for PrPres in Blood

R&D Objective Set-up a microplate Immunoassay for Proteinase K-resistant Prion (PrPres) detection in blood

Rationale Possible PrPsc oligomer precursors or re-circulating fragments, in blood and CSF. Detectable if captured and concentrated in ELISA- compatible final conditions.

4HPerron TSEAC 09 2006

Ligand selection: STREPTOMYCIN

Streptomycin binds and aggregatesprion proteins from brain SAF

0 5 10 20 0 5 10 20

Supernatant Precipitate

1 2 3 4 5 6 7 8 9 10 11 12

0 3.75m 7.5mM 15mM 30mM 120mM

Streptomycin Concentrations

Aly Moussa, Anthony W. Coleman, Anna Bencsik, Edwige Leclere, Florent Perret, Ambroise Martin and Hervé Perron. Chem. Commun., 2006, 973–975

5HPerron TSEAC 09 2006

Reticulation of PrP by Streptomycin

H2

O

H2O

H2O

H2O

H2O

H2O

PrPcAlpha-Helix

Streptomycin

PrP monomer

PrPscBeta-Sheet

6HPerron TSEAC 09 2006

Streptomycin protocol + WB Immunodetection: CJD Brain

SPECIFICITY: 100%SENSITIVITY: 100%

Without Ultracentrifugation !!

Human CJD Brain PrPres detection by WB after Streptomycin precipitation

I. Quadrio et al. Manuscript in preparation

52 non-CJD Dementia/Other Neuro. Dis. :All NEGATIVE

Alzheimer Disease 19, Lewy Body Dementia 4, Parkinson Disease 4, Fronto-Temporal Dementia 2, Vascular, ischaemic, metabolic 9, Others 14.

98 CJD patients: All POSITIVE : Sporadic : 80; Genetic : 14; Iatrogenic : 2; v-CJD : 2

7HPerron TSEAC 09 2006

Streptomycin-aggregated PrPres cannot be retained on Ab-coated microplates

Chemical denaturation is not compatible with antibody-coated microplates

Dilution of denaturing agent is not compatible with required sensitivity in blood

OHOH

OHOHOH OH

HO3SSO3HSO3H

HO3SSO3HSO3H

CALIX-ARENES: « molecular baskets » trapping macromolecular aggregates

Diluted HomogenateFrom BSE Brain

Sebastien Cecillon, Aly Moussa, Herve Perron, Anthony W. Coleman ChemComm. 2006 “in Press”

Compatiblewith capturein our drasticconditions

8HPerron TSEAC 09 2006

Coupling to solid phase

Present protocolPresent protocol

NHSNHS NHS NHS NHS NHS NHS NHS

NHS activated Microplate

NH2

Mono-Amino-

Calix-arene

Legend

(SO3H.CA)6(SO3H.CA)6-NH2-NH2

Chemical Coupling

NHS-NHS-Activated Activated surfacesurface

NH2NH2 NH2 NH2

S03H S03H S03H S03H S03H S03H S03H S03H

9HPerron TSEAC 09 2006

Test Principle Combining Streptomycin and Calix-Arenes

Plasma

Sample Buffer

Proteinase K

Immunodetection

PrecipitationBuffer

DenaturationBuffer

MicroplateCoupled to

Chemical Ligand

STREPTOMYCIN

CALIX-ARENESAnti-PrP

Monoclonal(s)

(BioMérieux)

Step 1: Step 1: Sample PreparationSample Preparation

Step 2: Step 2: Microplate ImmunoassayMicroplate Immunoassay

WASH

10HPerron TSEAC 09 2006

Human plasma « Pre-series »

0

100000

200000

300000

400000

500000

600000

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

CJD+Blood Donors

Cut-Off value is determined

for each experiment

with corresponding negative panel

0

500000

1000000

1500000

2000000

2500000

3000000

1 2 3 4 5 6 7 8 9

9716

9

8520

7

0791

7

3586

9

3660

3

3571

4

3009

4

3128

1

1161

6

1766

7

1265

3

8452

2

8207

0

PrP TM

Negative plasma Positive plasma Controls

Trial #1

1 mAb

Trial #5

2 mAb

CJD+Blood Donors T+/T-

11HPerron TSEAC 09 2006

Human Plasma (research lab prototypes)

*Samples sent for suspicion of CJD =« Atypical dementia ».

Trial #1

1 mAb

CJD

Patients

Blood

Donors

Dementia

Positive 17 0 (2) *

Negative 3 500 43Total 20 500 46

Trial #5

2 different mAb for Immunodetection

(CJD casesDifferent from Trial #1)

2 Genetic CJD

3 prob. Sporadic CJD

2 New-Variant CJD

2 Iatrogenic (GH) CJD

4 Def. Sporadic CJD

Trial #5

2 mAb

CJD

Patients

Blood

Donors

Positive 13 2 ** £

Negative 0 43

Total 13 ** Stopped for optimisations

£ Cause identified

Specif. 100%Sensib. 85 %

Specif. 95 %Sensib. 100 %

12HPerron TSEAC 09 2006

Bovine Samples: VLA BSE series

Heparin bovine plasma series (VLA R512)

0

200000

400000

600000

800000

1000000

1200000

1400000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

pk 80 dilution 1/ 5 cut-off negative samples

13HPerron TSEAC 09 2006

Bovine Blood

Normal (Non-VLA)

Cow Plasma

(Unexposed cattle)

BSE

Plasma

(VLA)

NEG 192 2*

POS 0 38

(95%)

Total 192 40

* 2 samples with 2 and 6 days delay before freezing

14HPerron TSEAC 09 2006

Conclusion

– Feasability of detection of PrPres in plasma shown

Future R&D activities: – Optimizing assay design and protocol (e.g.

monoclonal, sample volume)– Automation sample preparation– Industrialization (standardisation of microplate

production lots)