# Presentation: 498121

## Detection of PrPres in plasma

- H. Perron
- Presented by P van Driessche

## 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*

## 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.

## Ligand selection: STREPTOMYCIN

- Streptomycin binds and aggregates
- prion proteins from brain SAF

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

## Reticulation of PrP by Streptomycin

## Human CJD Brain PrPres detection by WB after Streptomycin precipitation

**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

## CALIX-ARENES: « molecular baskets » trapping macromolecular aggregates

-  **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**

**CALIX-ARENES: ****« molecular baskets » trapping macromolecular aggregates**

- Diluted Homogenate
- From BSE Brain

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

- Compatible
- with capture
- in our drastic
- conditions

## Coupling to solid phase

- Present protocol

_**Chemical Coupling**_

## Test Principle Combining Streptomycin and Calix-Arenes

_**Plasma **_

**Sample Buffer**

**Proteinase**
** ****K **

**Immunodetection**

**Precipitation**
**Buffer**

**Denaturation**
**Buffer**

**Microplate**
***Coupled to ***
***Chemical Ligand***

_***STREPTOMYCIN***_

_***CALIX-ARENES***_

***Anti-PrP Monoclonal(s)***
***(BioMérieux)***

- Step 1: _Sample Preparation_

- Step 2: _Microplate Immunoassay_

**WASH**

## Human plasma « Pre-series »

**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

- 97169

- 85207

- 07917

- 35869

- 36603

- 35714

- 30094

- 31281

- 11616

- 17667

- 12653

- 84522

- 82070

- PrP

- TM

**Negative plasma**

**Positive plasma**

**Controls**

- Trial #1
- 1 mAb

- Trial #5
- 2 mAb

**CJD+**

**Blood Donors**

**T+****/T-**

## 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 | 43 |
| Total | 20 | 500 | 46 |

**Trial #5**
**2 different mAb for Immunodetection**
***(CJD cases***
***Different 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 %*

## Bovine Samples: VLA BSE series

## 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

## 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)