Immuno-Real Time-PCR as a sensitive diagnostic tool: case of prion proteins. Virginie Ruelle and Benaissa ElMoualij. Center of Research on Prion Protein, University of Liège, Liège, Belgium Freising-Weihenstephan 26th-30th March 2007 1
1. Introduction A. Prion diseases : Types Bovine Spongiform Encephalopathy (BSE) Scrapie Creutzfeldt-Jakob No-infectious CJD Infectious Variant CJD (vcjd) Transmissible neurodegenerative disorders which affect the Central Nervous System (CNS) 2
1. Introduction B. Prion diseases : Symptoms Dementia Are you afraid of BSE? Me? No, I m I m a rabbit! 3
1. Introduction B. Prion diseases : Symptoms Dementia Locomotive disorders 4
1. Introduction B. Prion diseases : Symptoms Dementia Locomotive disorders Spongiform pathology of brain Vacuole Aggregate Fatal 5
1. Introduction C. Prion diseases : Infectious agent Normal protein : PrP c. Cellular prion. No-pathogenic. Soluble Abnormal protein : PrP sc ou PrP. Scrapie Prion. Pathogenic. Relatively no-soluble. Proteinase K resistant PrP res res Sequence = Folding 6
1. Introduction C. Prion diseases : Infectious agent PrP c 7
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 8
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 9
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 10
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 11
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 12
1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc Aggregate 13
1. Introduction D. Prion diseases : vcjd Contamination 14 Ludlam CA et al. British Journal of Haematology, 132, 13 24.
1. Introduction D. Prion diseases : vcjd Contamination Confirmed cases Cow BSE 188.417 From 1987 Contaminated Food Human vcjd 198 From 1995 15 Ludlam CA et al. British Journal of Haematology, 132, 13 24.
1. Introduction D. Prion diseases : vcjd Contamination Confirmed cases Cow BSE 188.417 From 1987 Contaminated Food 31 Blood donors 66 Blood recipients Human vcjd 198 From 1995 Contaminated Blood 4 From 2004 Diagnostic 17
1. Introduction D. Prion diseases : vcjd Contamination New sensitive test to detect prion : Say Meuh Pré-clinical phase (LONG) Clinical Phase (SHORT) No Meuh!! Inoculation Ingestion of BSE contaminated food First Death clinical signs Disease blocking 18 Bruce ME et al. Immunol Today. 2000;21(9):442-6.
1. Introduction E. Prion diseases : Diagnostic Living : Symptoms Post-mortem : BRAIN - Histological coupe - Molecular diagnostic 19
Rapid BSE testing kits approved by the European Commission ELISA BioRad TeSeE Detection Kit ENFER TSE kit v.2.0 InPro CDI-5 Institut Pourquier Speed it BSE Prionics Check LIA Roche Applied Science PrioScreen Roboscreen BetaPrion BSE EIA test kit Western Blot Other Prionics Check Western Ceditect BSE test Prionics Check PrioStrip IDEXX Herdchek BSE Antigen Test Kit EIA 20
1. Introduction E. Prion diseases : Molecular Diagnostic SENSITIVITY Classical assays on PrP sc Western blot ELISA Threshold of sensitivity 1000-3000 IU/mL ~ 1000 IU/mL Clinical phase : > 100 IU/mL immuno-pcr Pré- Clinical phase : < 10 IU/mL 21 Barletta JM et al. J. Virol. Methods. 2005;127:154-164.
2. Immuno-PCR A. Principle : Detection antibody DNA PCR Antigen Capture Antibody Signal amplification Plate 22
2. Immuno-PCR A. Principle : Biotinylated Detection antibody B Antigen Capture Antibody Plate 23
2. Immuno-PCR A. Principle : Biotinylated Detection antibody B Strep Antigen Capture Antibody Plate 24
2. Immuno-PCR A. Principle : Chimeric DNA Nature Matrix Biotinylated Detection antibody B Strep Antigen Capture Antibody Plate Patent: EP1232283,2002-08-21. i-detect 25Company www.i-detect.eu
2. Immuno-PCR A. Principle : Nature Chimeric DNA Organisms Matrix A B C Synthetic Patent: EP1232283,2002-08-21. i-detect 26Company www.i-detect.eu
2. Immuno-PCR No-pathogenic B. Evaluation on human recombinant prion protein 4F7 PrP rec 1E5 B Strep Plate ELISA Detection limit ~ 10 ng/ml immuno-pcr Detection limit ~ 100pg/mL Improvement 100 x Cycle t 24 25 26 27 28 29 30 ipcrq 100 pg/ml Recombinante Prion Detection limit ELISA 10 ng/ml 31 0 0 1 2 3 4 5 6 7 Log C 2 1 Optical Density 27
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 28
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Studied cases Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 29
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Studied cases Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 30
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Sample preparation Plate Middle frontal gyrus Diagnostic surgical biopsies Have been performed previously Homogenization PK digestion Concentration Dilution 31 Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) 24 25 26 27 28 29 30 31 Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x 2.0 1.5 1.0 0.5 32 0.0 1 10 100 1000 Dilution (1/x) Optical Density (450nm) Cycle threshold (Ct) 24 25 26 27 28 29 30 31 Prion in brain Control n 1 32 1 10 100 1000 0.0 Dilution (1/x) 2.0 1.5 1.0 0.5 Optical Density (450nm) 4F7 B Strep Improvement 50 x PrP rec 1E5 Plate Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 32
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) 24 25 26 27 28 29 30 31 Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x 2.0 1.5 1.0 0.5 32 0.0 1 10 100 1000 Dilution (1/x) Optical Density (450nm) Cycle threshold (Ct) Prion in brain Control n 1 to 7 24 2.0 25 26 27 1.5 28 29 30 1.0 31 32 33 0.5 34 35 36 1 10 100 1000 0.0 Dilution (1/x) Optical Density (450nm) 4F7 B Strep Improvement 50 x PrP rec 1E5 Plate Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 33
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) 24 25 26 27 28 29 30 31 Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x 2.0 1.5 1.0 0.5 32 0.0 1 10 100 1000 Dilution (1/x) Optical Density (450nm) 4F7 B Strep Improvement 50 x PrP rec 1E5 Plate Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 34
2. Immuno-PCR Pathogenic Cycle threshold (Ct) Cycle threshold (Ct) Cycle threshold (Ct) Prion in brain CJD n 2 24 2.0 25 26 1.5 27 28 ipcrq 5.000x 1.0 29 30 ELISA 100x 0.5 31 32 0.0 1 10 100 1000 Dilution (1/x) Prion in brain CJD n 3 24 2.0 25 26 1.5 27 ipcrq 28 1.000x 1.0 29 30 31 32 ELISA 10x 0.5 0.0 1 10 100 1000 Dilution (1/x) Prion in brain CJD n 4 24 2.0 25 26 1.5 27 28 ipcrq 500x 1.0 29 ELISA 30 31 32 5x 0.5 0.0 1 10 100 1000 Dilution (1/x) C. Application : diagnostic of Creutzfeldt-Jakob Disease Validation 50 x 100 x 100 x 100 x ( ) ( ) ( ) Prion in brain CJD n 5 24 2.0 25 26 1.5 27 28 1.0 29 30 31 32 ipcrq ELISA 5.000x 50x 0.5 0.0 1 10 100 1000 Dilution (1/x) Prion in brain CJD n 6 24 2.0 25 26 1.5 27 28 ipcrq 1.000x 1.0 29 30 31 32 ELISA 10x 0.5 0.0 1 10 100 1000 Dilution (1/x) Prion in brain CJD n 7 100 x 20 x 24 2.0 25 26 1.5 27 28 29 ipcrq 1.000x 1.0 30 ELISA 50x 0.5 31 32 0.0 1 10 100 1000 Dilution (1/x) Optical Density (450nm) Optical Density (450nm) Optical Density (450nm) Cycle threshold (Ct) 24 25 26 27 28 29 30 31 Prion in brain CJD n 1 to 7 2.0 1.5 1.0 0.5 32 0.0 1 10 100 1000 Improvement 20 to 100 x 35 Optical Density (450nm) Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11.
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Validation CJD Controls rec 1 2 3 4 5 6 7 1 2 3 4 5 6 7 75 50 37 25 20 10 Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 36
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease 75 50 37 25 20 10 Validation CJD Controls rec 1 2 3 4 5 6 7 1 2 24 3 4 5 6 7 Cycle threshold (Ct) 25 26 27 28 29 30 31 Prion in brain CJD n 4 ELISA 5x 2.0 1.5 1.0 0.5 32 0.0 1 10 100 1000 Dilution (1/x) 100 x ipcrq 500x Gofflot S et al. Clinical chemistry. 2005;51(9):1605-11. 37
2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease. Comparison of brain samples 7 patients with sporadic CJD / 7 controls using : ELISA Western-Blot immuno-pcr. All methods shows similar sensitivity and specificity (100% detected) Signal improvement compared to ELISA and Western-Blot ~ 100x on pathogenic protein (CJD). immuno-pcr as a new useful tool for ante-mortem PrPres detection 38
2. Immuno-PCR D. Protocol improvement vs reproducibility: Buffer/Antibodies/DNA/Plate controls Strep Plate Detection complexes 4F7 BStrep Plate 4F7 biotinylated39antibody Roboscreen
Background 8 controls Day n 1 1 at Ct=38 7 at Ct=40 Day n 2 1 at Ct=38,5 7 at Ct=40 Day n 3 1 > Ct=37 7 at Ct=40 40
Reproducibility 8 detection complexes Day n 1 8 Ct in [25-26] Day n 2 8 Ct in [25-26] Day n 3 8 Ct in [25-26,2] 41
2. Immuno-PCR E. Detection in blood: EUROPE 6 e Framework Programme 2007-2010 EUDICA LANCASTER UNIVERSITY Plasma lab Jozef Stefan Institute Ljubljana CHU Lyon National Institute of Psychiatry and Neurology BUDAPEST 42
2. Immuno-PCR E. Detection in blood: EUROPE 6 e Framework Programme 2007-2010 LANCASTER UNIVERSITY Plasma lab Jozef Stefan Institute Ljubljana CHU Lyon Diagnostic kits Creutzfeldt-Jakob Alzheimer Parkinson EUDICA National Institute of Psychiatry and Neurology BUDAPEST 43
3. Conclusions. Immuno-PCR as detection tool could be used to detect pathogenic and no-pathogenic forms of Prion protein (and others). Immuno-PCR as ultrasensitive tool Signal improvement compared to ELISA - 100x on pathogenic protein (CJD). Immuno-PCR with low background Buffers/Antibodies/DNA/plate improvement - Background near of a Ct= 40 - Reproductibility intra and inter-days. immuno-pcr as a new useful for ante-mortem PrPres detection IN BLOOD 44
Acknowledgements 45
Acknowledgements EUDICA LANCASTER UNIVERSITY Plasma lab Jozef Stefan Institute Ljubljana CHU Lyon National Institute of Psychiatry and Neurology BUDAPEST 46
Thank you for your attention Contacts : v.ruelle@ulg.ac.be www.i-detect.eu 47