Immuno-Real Time-PCR as a sensitive diagnostic tool: case of prion proteins.

Transcription

1 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

2 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

3 1. Introduction B. Prion diseases : Symptoms Dementia Are you afraid of BSE? Me? No, I m I m a rabbit! 3

4 1. Introduction B. Prion diseases : Symptoms Dementia Locomotive disorders 4

5 1. Introduction B. Prion diseases : Symptoms Dementia Locomotive disorders Spongiform pathology of brain Vacuole Aggregate Fatal 5

6 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

7 1. Introduction C. Prion diseases : Infectious agent PrP c 7

8 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 8

9 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 9

10 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 10

11 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 11

12 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc 12

13 1. Introduction C. Prion diseases : Infectious agent PrP c PrP sc Aggregate 13

14 1. Introduction D. Prion diseases : vcjd Contamination 14 Ludlam CA et al. British Journal of Haematology, 132,

15 1. Introduction D. Prion diseases : vcjd Contamination Confirmed cases Cow BSE From 1987 Contaminated Food Human vcjd 198 From Ludlam CA et al. British Journal of Haematology, 132,

16 1. Introduction D. Prion diseases : vcjd Contamination Confirmed cases Cow BSE From 1987 Contaminated Food 31 Blood donors 66 Blood recipients Human vcjd 198 From 1995 Contaminated Blood 4 From 2004 Diagnostic 17

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.

18 1. Introduction E. Prion diseases : Diagnostic Living : Symptoms Post-mortem : BRAIN - Histological coupe - Molecular diagnostic 19

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

20 1. Introduction E. Prion diseases : Molecular Diagnostic SENSITIVITY Classical assays on PrP sc Western blot ELISA Threshold of sensitivity 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:

21 2. Immuno-PCR A. Principle : Detection antibody DNA PCR Antigen Capture Antibody Signal amplification Plate 22

22 2. Immuno-PCR A. Principle : Biotinylated Detection antibody B Antigen Capture Antibody Plate 23

23 2. Immuno-PCR A. Principle : Biotinylated Detection antibody B Strep Antigen Capture Antibody Plate 24

24 2. Immuno-PCR A. Principle : Chimeric DNA Nature Matrix Biotinylated Detection antibody B Strep Antigen Capture Antibody Plate Patent: EP , i-detect 25Company

25 2. Immuno-PCR A. Principle : Nature Chimeric DNA Organisms Matrix A B C Synthetic Patent: EP , i-detect 26Company

26 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 ipcrq 100 pg/ml Recombinante Prion Detection limit ELISA 10 ng/ml Log C 2 1 Optical Density 27

27 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Gofflot S et al. Clinical chemistry. 2005;51(9):

28 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Studied cases Gofflot S et al. Clinical chemistry. 2005;51(9):

29 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Studied cases Gofflot S et al. Clinical chemistry. 2005;51(9):

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):

31 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x Dilution (1/x) Optical Density (450nm) Cycle threshold (Ct) Prion in brain Control n 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):

32 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x Dilution (1/x) Optical Density (450nm) Cycle threshold (Ct) Prion in brain Control n 1 to 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):

33 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease ELISA/iqPCR comparison Cycle threshold (Ct) Prion in brain CJD n 1 ELISA 100x ipcrq 5.000x 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):

34 2. Immuno-PCR Pathogenic Cycle threshold (Ct) Cycle threshold (Ct) Cycle threshold (Ct) Prion in brain CJD n ipcrq 5.000x ELISA 100x Dilution (1/x) Prion in brain CJD n ipcrq x ELISA 10x Dilution (1/x) Prion in brain CJD n ipcrq 500x ELISA x Dilution (1/x) C. Application : diagnostic of Creutzfeldt-Jakob Disease Validation 50 x 100 x 100 x 100 x ( ) ( ) ( ) Prion in brain CJD n ipcrq ELISA 5.000x 50x Dilution (1/x) Prion in brain CJD n ipcrq 1.000x ELISA 10x Dilution (1/x) Prion in brain CJD n x 20 x ipcrq 1.000x ELISA 50x Dilution (1/x) Optical Density (450nm) Optical Density (450nm) Optical Density (450nm) Cycle threshold (Ct) Prion in brain CJD n 1 to Improvement 20 to 100 x 35 Optical Density (450nm) Gofflot S et al. Clinical chemistry. 2005;51(9):

35 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Validation CJD Controls rec Gofflot S et al. Clinical chemistry. 2005;51(9):

36 2. Immuno-PCR Pathogenic C. Application : diagnostic of Creutzfeldt-Jakob Disease Validation CJD Controls rec Cycle threshold (Ct) Prion in brain CJD n 4 ELISA 5x Dilution (1/x) 100 x ipcrq 500x Gofflot S et al. Clinical chemistry. 2005;51(9):

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

38 2. Immuno-PCR D. Protocol improvement vs reproducibility: Buffer/Antibodies/DNA/Plate controls Strep Plate Detection complexes 4F7 BStrep Plate 4F7 biotinylated39antibody Roboscreen

39 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

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

41 2. Immuno-PCR E. Detection in blood: EUROPE 6 e Framework Programme EUDICA LANCASTER UNIVERSITY Plasma lab Jozef Stefan Institute Ljubljana CHU Lyon National Institute of Psychiatry and Neurology BUDAPEST 42

42 2. Immuno-PCR E. Detection in blood: EUROPE 6 e Framework Programme 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

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

44 Acknowledgements 45

45 Acknowledgements EUDICA LANCASTER UNIVERSITY Plasma lab Jozef Stefan Institute Ljubljana CHU Lyon National Institute of Psychiatry and Neurology BUDAPEST 46

46 Thank you for your attention Contacts : v.ruelle@ulg.ac.be 47