Use of PMCA for Biochemical Diagnosis of Prion Diseases Claudio Soto, PhD Dept of Neurology, University of Texas Medical Branch and Amprion Inc.

Importance of Prion Diagnosis Food industry Blood banks Plasma products Disease diagnosis Clinical trials Brain surgery Drugs from human origin Organ transplant Soto (2005) Nature Rev Microbiol. 2:

PrP sc Is the most specific marker for the disease. However, its levels in body fluids and tissues other than nervous system are too low to be detected Design a more sensitive test for PrP sc detection or Amplify the level of the marker (PrP Sc ) The problem of Prion Diagnosis

d y Clinical symptoms y PrP C Slow process PrP Sc Infection Incubation Time Disease PrP Sc amplification during disease propagation

PrP C Incubation Growing of units Incubation Growing of units + Protein Misfolding Cyclic Amplification (PMCA) PrP Sc Soto et al. (2002) Trends Neurosci. 25: Sonication Multiplication of units Sonication Multiplication of units

Brain homogenates Healthy hamster Sick hamster (263k) PK digestion WB analysis Amplification (Incubation + sonication) PMCA: Proof of concept source of PrP Sc source of PrP c PrP res fragment PK + + PMCA - + Saborio, Permanne and Soto (2001) Nature 411:

One-by-one 96 format control Amplified Non Amplified Automated PMCA

Ultrasensitive detection of PrP Sc by serial PMCA 1.0x10 3 Dilution Non amplified Amplification factor ~ 6600 fold 3.0x x x x x x x x10 6 NBH -PK 2.0x x PMCA cycles 1:10 1 st PMCA (96 cycles) 2 nd PMCA (118 cycles) 2.5x10 4 Dilution 1.3x x x10 6 Dilution 3.1x x x x x x x x x10 3 NBH -PK NBH -PK 2.5x x10 12 Non amplified Amplification factor ~10 million folds Castilla, Saa and Soto (2005) Nature Medicine 11:

NBH -PK Scrapie LD50 spiked 1 st PMCA (144 cycles) 2 nd PMCA (144 cycles) 3 rd PMCA (144 cycles) 4 th PMCA (144 cycles) Non amplified What is the minimum quantity detected? 4 th PMCA (144 cycles) Scrapie LD50 spiked 7 th PMCA Our mathematical estimation is that LD50 contains around molecules of PrP monomer Saa, Castilla, and Soto (2006) J. Biol. Chem (In press)

Sensitivity of PrP Sc detection by different methods 7 rounds PMCA 1.0 x ag 26 3,300,000,000 1 round PMCA 2 rounds PMCA Saa, Castilla, and Soto (2006) J. Biol. Chem (In press)

Symptomatic phase Plasma0.1 pg/ml = 2 x 10 7 molecules Buffy coat1 pg/ml = 2 x 10 8 molecules Incubation period (pre-symptomatic phase) Plasma pg/ml = 1-2 x 10 6 molecules Buffy coat pg/ml = 1-2 x 10 7 molecules Can we detect PrP Sc in blood? How much PrPSc is there in blood? Our sensitivity Single PMCA: 65 pg/ml = 1.3 x 10 9 molecules/ml Double PMCA: 0.02 pg/ml = 4 x 10 5 molecules/ml 7 th rounds PMCA: fg/ml = 500 molecules/ml Taken from Brown et al (2001) J. Lab Clin. Invest. 137: 5-13 In other words sensitivity of PrP Sc detection as compared with standard western blot has to be increase by 100,000 – 1,000,000 fold

Can we detect PrP Sc in blood? Castilla, Saa and Soto (2005) Nature Medicine 11:

Pre-symptomatic detection of PrP Sc in blood Blood taken during incubation period Infection Clinical disease 14 days I1 C1 I2 C2 I3 C3 I4 C4 I5 C5 NBH -PK /5 20 days40 days60 days70 days80 daysSymptomatic 3/6 I1 I2 I3 I4 I5 I6 C1 C2 C3 C4 NBH -PK /10 I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 C1 C2 C3 C4 C5 NBH -PK /5 I1 I2 I3 I4 I5 C1 C2 C3 C4 NBH -PK /5 C1 I1 C2 I2 C3 I3 C4 I4 C5 I5 NBH -PK /5 C1 I1 C2 I2 C3 I3 C4 I4 C5 I5 NBH -PK /10 C1 I1 C2 I2 C3 I3 C4 I4 C5 I5 C6 I6 C7 I7 C8 I8 C9 I9 C10 I10 NBH -PK Saa, Castilla and Soto (2006) Science 313:92-94

Leakage from the brain? Peripheral prion replication? Symptomatic phase Pre-symptomatic detection of PrP Sc in blood Saa, Castilla and Soto (2006) Science 313:92-94

Application of PMCA to different samples sCJD type 1 sCJD type 2 vCJD

What is next? Adapt and optimize blood detection of PrP Sc in relevant natural samples (cow, human, sheep, deer) Large scale study to evaluate the detection of PrP Sc in blood of healthy donors in countries with high risk of vCJD (UK, France, etc) Study earliest time in which PrP Sc can be detected in humans (primate model, familial cases) and in cattle (experimental infection model) Optimize the method for detection in other blood components (plasma, red cells) and other biological fluids (urine, CSF). Develop the technology into a high throughput and practical test

Joaquín Castilla, PhD Rodrigo Morales Paula Saá June Yowtak Lisbell Estrada Jorge de Castro Becky Daniels Claudio Soto, PhD Karim Abid, PhD

Gabriela Saborio, MD Celine Adessi, PhD Kinsey Maundrell, PhD Bruno Permanne, PhD Youcef Fezoui, PhD Raphaele Buser, PhD Milene Russelaskis, PhD Claudio Hetz, PhD Sergio Benavent Laurence Anderes Marie-Jose Frossard Santiago Fraga Elizabeth Vial Sergio Peano, MD Thomas Ruckle, PhD Former lab members Case Western Reserve University Pierluigi Gambetti CJD Unit, Edinburgh, UK Robert Will James Ironside Istituto Carlo Besta, Italy Fabrizio Tagliavini US Department of Agriculture Juergen Richt Istituto Superiore di Sanita, Italy Maurizio Pocchiari University of Kentucky Glenn Telling University of Edinburgh, UK Jean Manson University of Zurich, Switzerland Adriano Aguzzi Mathias Heikenwalder Collaborators