larry stanker and john mark carter agricultural research...
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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
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
Gibson et al., PNAS 97 5802 (2000)
[X][X]
O
OBS3
Surface Mapping by Distance Constraints
Bidentate Ligand
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
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
• 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