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)
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