Chester Sarrel ©

Prion Detection: Strategies and Challenges

Larry Stanker and John Mark CarterAgricultural Research ServiceWestern Regional Research CenterAlbany, CA USA

E.U.-U.S. Task Force on Biotechnology Research"Predictive, Forward Thinking on Emerging Infectious Diseases"June 21-22nd in Brussels

Transmissible Spongiform Encephalopathies (TSE) or prion diseases

• Slow, transmissible brain diseases• Occurs in a variety of mammals including humans• After infection, disease can take from months to decades to appear• Infections can occur from ingestion or inoculation• Always fatal

No sensitive, pre-clinical diagnostic test availableNo effective pre- or post-clinical treatments available

• The infectious agent is thought to be a prionExact nature of prions and mechanism of action is not clearly understood

Scrapie 1755 (1947 US)

Transmissible Mink Encephalopathy (TME) 1947

Bovine Spongiform Encephalopathy (BSE) 1986Feline Spongiform

Encephalopathy (FSE) 1990

Chronic Wasting Disease (CWD) 1967

. Source: E. Williams,University of Wyoming (May, 2003)

TSE’s in Animals

• Creutzfeldt-Jakob disease• Gerstmann-Strausslar Scheinkar syndrome• Fatal familial insomnia• Kuru• Iatrogenic CJD• New variant CJD (nvCJD)

Human TSE Diseases

Non infectious Infectious

Prion Detection

•Histological

Coarse spongiform degeneration and PrP immunoreactivity, often located at the rim of the vacuoles or forming plaque-like aggregate occasionally surrounded by vacuoles in the cerebral cortex

Source: Sporadic and familial CJD: classification and characterisation, by Pierluigi Gambetti, Qingzhong Kong, WenquanZou, Piero Parchi, and Shu G. Chen British Medical Bulletin, Volume 66, 2003 p 213-239.

Prion Detection

•Histological •Immunological picomolar (1pmol=40ng)

–IHC

Coarse spongiform degeneration and PrP immunoreactivity, often located at the rim of the vacuoles or forming plaque-like aggregate occasionally surrounded by vacuoles in the cerebral cortex

Source: Sporadic and familial CJD: classification and characterisation, by Pierluigi Gambetti, Qingzhong Kong, WenquanZou, Piero Parchi, and Shu G. Chen British Medical Bulletin, Volume 66, 2003 p 213-239.

Prion Detection

•Histological •Immunological picomolar (1pmol=40ng)

–IHC

–Western Blots

Source: Biochemistry and structure of PrPC and PrPSc, Detlev Riesner, inBritish Medical Bulletin, Volume 66, 2003 p 21-33.

–ELISA

–Conformation Dependent Immunoassay (CDI)

Conformation Dependent Immunoassay (CDI)

Eu

PrPC PrPC PrPC

Capture

1. Native Rx

PrPC

2. Denatured Rx

Wash

+

2nd AbPrPC PrPC PrPC

PrPSc PrPSc PrPSc Eu Eu

Reporter Antibody

PrPC

Native

Wash

+

2nd Ab

Eu Eu EuEuEuEu

PrPC

PrPS

c

Denatured

mAb epitopes (residues ~90 - 125) buried in native PrPSc

PrPC

PrPSc

Capture

PrPScPrPSc

aCDI Analysis of BSE Positive vs Normal Brains

1

10

100

1000

10000

100000

0 10 20 30 40 50 60 70 80

Sample Number

PrP

tota

l-

PrP

C(D

-N)

BSE

Normal

•Instrumental

-Mass Spectrometric (attomolar)

Prion Detection•Histological•Immunological picomolar (1pm=40ng)

–IHC–Western Blots

–ELISA

–Conformation Dependent Immunoassay (CDI)

•Bioassay

Certified Assays for BSE

• Western Blot Test - Prionics AG Switzerland

• ELISA - Enfer Inc. Ireland

• ELISA - Bio-Rad, France

• Luminescent Immunoassay (LIA) - Prionics AG Switzerland

• Conformation Dependent Immunoassay (CDI) -Inpro, USA

0/2010-3.5

1/2010-3.0

18/2010-2.5

20/200/2010-2.0

20/2020/200/2010-1.5

20/2020/2015/20 (+2?)b10-1

6/66/66/60

Bio-Rad Inc.ELISA

Enfer ScientificELISA

PrionicsCheck Western

Estimated Detection Limits of the First ThreeEC-Approved Postmortem Tests for BSE

Dilution of Homogenate

Number of BSE-infected Brain-Homogenate Samples ScoringPositive

NOTE: The data represent the number of samples testing positive/total number of samples tested.a … positive brain homogenate of known infectivity titer was tested at dilutions in negative brain (Moynagh & Schimmel, 1999:105B Two samples rated inconclusive at this dilution.

SOURCE adapted from Moynagh & Schimmel (1999) as reported in Advancing Prion Science, Inst. Medicine, Nat Acad. Sci USA 2004.

Conformational Dependent Immunoassay

Safar, J., H. Wille, V. Itri, D. Groth, H. Serban, M. Torchia, F. E. Cohen and S. B. Prusiner(1998). “Eight prion strains have PrPSc molecules with different conformations.” Nat. Med. 4(10):1157-1165

Instrumental

Protein Misfolding Cyclic AmplificationSaborio et al. 2001

PK Sensitivity

yes 10-100 times > thanWestern blotting

Capillary Gel Electrophoresis yes 100 fold > sensitivitythan western blotting

Fluorescence Correlation Spectroscopy no2 pM 20 times moreSensitive than Western blotting

Multispectral Ultraviolet Fluorescence yes in pM range

Fourier Transform Infrared Spectroscopy(FTIR)

no ?

Source: Nat. Acad. Sci 2003

Challenges

• All Postmortem• Sensitivity in Pico mole • Extensive Sample Preparation• Not applicable to environmental

sampling• Do not distinguish strains

Mass Spectrometry

Gibson et al., PNAS 97 5802 (2000)

[X][X]

O

OBS3

Surface Mapping by Distance Constraints

Bidentate Ligand

3-dimensional view of normal PrPc protein with reactive Lysine residues in red

185

194

204

220

Mass, amu0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3397

Full length PrPC

Experimental Mass Theoretical mass16,240.6 16,241.5

rshPrP Hamster PrPC 90-231

Mass, amu0

50

100

150

200

250

300

350

400

450

50016,239.56

Derivatized Hamster PrPC

Intact PrP

Partial derivatized PrP

Fully derivatized

MS Progress

• rPrPDerivatization completed, identification of derivatized peptides in progress

• PrPC

Isolation in progress• PrPSc

12 derivative peptides tentatively identifiedDimeric cross-links not observed in rPrP

TagTag

Tag Tag Tag

Double Sandwich ELISA

Capture antibody

Primary antibody

PrP protein

Reporter antibody

Improvement to CDI

• Improved sensitivity with better monoclonals

• Strain typing• Format

• Species identification

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

F-4 31/23F-4 31/23

36

29

22

88

51

PrP

P

rP 0/

00/

0

Mo

Mo

SH

aS

Ha

Hu

Hu

Bo

Bo

recB

o re

cBo

Ov

Ov

M D

M DKd

Sandwich ELISA: Titration of purified 20D7(Prot.G) & M3 on rHu 90-231 and rBo 23-231

100

1,000

10,000

100,000

1,000,000

510.20.040.0080.0023E-046E-051E-053E-065E-071E-07

concentration (µg/ml)

20D7 Prot. G/rHu90-23120D7 Prot. G/rBo 23-231M3 /rHu90-231M3/rBo90-231

Future Challenges• Complete the MS method• Apply the MS method to strain differentiation• New antibodies to improve the CDI• Develop new mouse lines for antibody production• Develop live animal test

Knowledge Gaps

• Live animal testImproved sensitivityIdentify best tissue source

• In-vitro Amplification Method• Strain Differentiation (physical methods)• Improved sample preparation/concentration

methods• Identify alternative tissue• Identify optimum sample time

Acknowledgements / Collaborators

WRRCChris SilvaBruce OniskoPaul Merrill

Univ. Santiago Santiago, Spain

Jesús Reqúena

UCSF - Institute for Degenerative Disease

Jiri SafarAna SerbanStanley Prusiner

ARS Pullman WAAmes IA

U.S. Beef Production Totals (billion pounds)US Total

1999 26,3872000 26,7762001 26,1022002 27,0892003* 26,296* Estimate based on year-to-date

U.S. Beef Exports During the first 10 months of 2003 beef exports represented 9.6% of domestic production (2.17 billion pounds vs. 22.5 billion pounds) During 2002, beef exports represented 9% of domestic production (2.45 billion pounds vs. 27.1 billion pounds). Beef exports, during 2003 year-to-date, have been worth $2.664 billion, variety meat exports have been worth $601 million and tallow exports have been worth $325 million. During 2002, beef exports were valued at $2.585 billion, beef variety meats were valued at $618 million and tallow at $343 million for a total value of $3.55 billion.

U.S. Beef Exports (000 pounds carcass weight)Japan Korea Mexico Canada Other US Total

1999 1,101,166 307,957 466,001 249,463 292,528 2,417,115 2000 1,116,855 398,825 533,616 253,694 213,281 2,516,271 2001 1,004,975 345,638 532,134 233,271 154,709 2,270,727 2002 784,325 597,258 629,042 240,543 196,155 2,447,323 2003* 878,444 610,908 621,459 245,293 302,110 2,658,215

* Estimate based on year-to-dateJanuary 5, 2004Source: CattleFax & National Cattlemen’s Beef Association

Percentage of U.S. beef exports to countries% of Exports Japan Korea Mexico Canada Other1999 46% 13% 19% 10% 12%2000 44% 16% 21% 10% 8%2001 44% 15% 23% 10% 7%2002 32% 24% 26% 10% 8%2003* 33% 23% 23% 9% 11%* Estimate based on year-to-date

Value of beef, beef variety meat and tallow exports (billion dollars)

Beef Variety meats Tallow Total1999 2.724 .557 .372 $ 3.6532000 2.987 .628 .261 $ 3.8752001 2.633 .772 .237 $ 3.6422002 2.586 .619 .343 $ 3.5492003* 2.665 .601 .325 $ 3.591

* 2003 YTD through October

January 5, 2004Source: CattleFax & National Cattlemen’s Beef Association

3-dimensional view of normal PrPc protein with reactive Lysine residues in red

LYS-LYS distance

† 185 to 204 17.2 Å

† 185 to 194 18.7 Å

194 to 204 21.7 Å

195 to 220 27.5 Å

185 to 220 30.2 Å

204 to 220 29.9 Å

185

194

204

220

†crosslinks predicted

Immunochemistry

U.S. Beef ExportsDuring the first 10 months of 2003 beef exports represented 9.6% of domestic production (2.17 billion pounds vs. 22.5 billion pounds) During 2002, beef exports represented 9% of domestic production (2.45 billion pounds vs. 27.1 billion pounds). Beef exports, during 2003 year-to-date, have been worth $2.664 billion, variety meat exports have been worth $601 million and tallow exports have been worth $325 million. During 2002, beef exports were valued at $2.585 billion, beef variety meats were valued at $618 million and tallow at $343 million for a total value of $3.55 billion.

U.S. Beef Exports (000 pounds carcass weight)Japan Korea Mexico Canada Other US Total

1999 1,101,166 307,957 466,001 249,463 292,528 2,417,115 2000 1,116,855 398,825 533,616 253,694 213,281 2,516,271 2001 1,004,975 345,638 532,134 233,271 154,709 2,270,727 2002 784,325 597,258 629,042 240,543 196,155 2,447,323 2003* 878,444 610,908 621,459 245,293 302,110 2,658,215

* Estimate based on year-to-date

January 5, 2004Source: CattleFax & National Cattlemen’s Beef Association

Value of beef, beef variety meat and tallow exports (billion dollars)

Beef Variety meats Tallow Total1999 2.724 .557 .372 $ 3.6532000 2.987 .628 .261 $ 3.8752001 2.633 .772 .237 $ 3.6422002 2.586 .619 .343 $ 3.5492003* 2.665 .601 .325 $ 3.591

* 2003 YTD through October

Percentage of U.S. beef exports to countries% of Exports Japan Korea Mexico Canada Other1999 46% 13% 19% 10% 12%2000 44% 16% 21% 10% 8%2001 44% 15% 23% 10% 7%2002 32% 24% 26% 10% 8%2003* 33% 23% 23% 9% 11%* Estimate based on year-to-date

Value of beef, beef variety meat and tallow exports (billion dollars)Beef Variety meats Tallow Total

1999 2.724 .557 .372 $ 3.6532000 2.987 .628 .261 $ 3.8752001 2.633 .772 .237 $ 3.6422002 2.586 .619 .343 $ 3.5492003* 2.665 .601 .325 $ 3.591

* 2003 YTD through October

January 5, 2004Source: CattleFax & National Cattlemen’s Beef Association

TSE Studies Underway

Detection of meat and bone meal in animal feedMonoclonal antibodies to PrPPrion detection by mass spectrometry

20D7 Different Antigen Levels Antigen PrP

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.01 0.1 1 10

antibody dilutio

10

5

2

1

0

20D7 Different Antibody Levels Antigen is

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12

antigen dilution µg/

1

0.5

0.25

0.125

0.0625

0.03125

0.015625

M3 Different Antigen LevAntigen is rHu PrP

00.5

11.5

2

2.2

1.1

0.55

0.27

50.

1375

0.06

875

0.03

438

Antibody concentratµg/mL

10

5

2

1

20D7 Different Antibody levels antrHu PrP

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

10521

Antigen concentation µg

2.2

1.1

0.55

0.275

0.1375

0.06875

0.034375

Double Sandwich ELISA - Antigen Optimization

Meat and Bone Meal

• Stable to rendering conditions• Indicative of animal materials• High concentration in brain and offal• Trace amounts in plants

Bone Meal

Feed Blood Meal

Cotton SeedMeal

HOHO

Meat and Bone Meal - Major Route of BSE Infection (UK)

• Canada & US Implemented Ban 1997• Analytical Methods Lacking• Sterols Represent Useful Markers

Cholesterol> 90 different additives used

A

B

MS of soybean meal without (A) and with (B) 1% added MBM

• Developed a GC/MS Based Method• Evaluating > 90 Feed Additives

Future Studies• Immunoassay• Fatty Acids• Peptides

Cholesterol

Cholesterol

TIC

386

301

TIC

386

301

Material Cholesterol (mg/kg)

Bone meal 780MBM 430Canola meal < 1Linseed meal < 1Cotton seed meal 1Soybean meal < 120 % Corn gluten < 1Cotton seed hulls < 1

HOHO