Using biomarkers to monitor hormone abuse

Transcription

1 Using biomarkers to monitor hormone abuse Jeroen van Meeuwen Veterinary Public Health Division Institute for Risk Assessment Sciences CRL workshop 23 Okt 2008 Utrecht Utrecht University

2 OVERVIEW of PRESENTATION 2/36 Biomarkers BioCop Results: identification SPR prototype profiling Take-home messages

3 What is in the green box? (Bio)Markers 3/36

4 Multi-Endpoint 4/36 cell effector 1 Receptor = analyte Responses Nucleus Cytoplasm

5 Multi-Endpoint 5/36 cell effector 2 = analyte Receptor Nucleus Responses Cytoplasm

6 Multi-Endpoint 6/36 cell effector 3 = analyte Receptor Nucleus Responses Cytoplasm

7 Tools 7/36 transcriptomics proteomics microarrays molecular immunology Urine Blood Interstitial fluid Hair. biosensor technology metabolomics mass-spectrometry biological activity-screen bioprepology bioinformatics The world s finest collection of -ologies & -omics

8 Proteomics 8/36 Proteome: entire complement of proteins varies per circumstance Proteomics: any study that investigates these proteins

9 What is a biomarker? 9/36 Biomarker Indicator molecule for a certain response to a change in environment Proteomic & Biomarker: The indicator molecule is a protein or peptide. Well-chosen indicator proteins reflects an environment change fingerprint: panel of BMs changes in response to a certain signal.

10 Dynamic equilibrium: animal as multi-endpoint 10/36 Plant protein Animal protein anabolics GROWTH & MAINTENANCE & REPRODUCTION catabolism anabolism catabolism anabolism catabolism anabolism ENERGY cf. use of biomarkers in oncology, clinical chemistry and pathology

11 Detection of anabolic residues FAILS 11/36 approx. 10% of European cattle is illegally treated with anabolics (estimate of authorities!) # found non-compliant at official inspection: average 0.3%! Gap between reality and findings

12 Recent abuse of anabolics 12/36 AID vindt groeibevorderaar testosteron bij runderen De Algemene Inspectiedienst (AID) van het ministerie van LNV heeft gericht monsters genomen bij 22 runderen die waren gehuisvest bij een Nederlandse veehouder. Op zijn bedrijf stonden 170 runderen van een Belgische eigenaar. De monsters zijn drie weken geleden genomen. Uit de laboratoriumresultaten die deze week bekend werden, blijkt nu dat 15 dieren positief zijn getest op de verboden groeibevorderaar Testosteron. De veehouder heeft in de weken na de bemonstering alle 170 runderen versneld laten slachten. De Voedsel en Waren Autoriteit (VWA) is op basis van de bevindingen bezig de karkassen en het nog niet geconsumeerde vlees van de positief geteste dieren terug te halen. De 170 runderen die bij de Nederlandse veehouder stonden, waren van een Belgische handelaar. De dieren werden hier enkele maanden geleden ondergebracht en verder gemest, voordat ze naar het slachthuis gaan. De AID kwam drie weken geleden bij deze veehouder na uitgebreide analyses van ongebruikelijke transportbewegingen van slachtvee, zowel in Nederland als bij import en export. Samen met de VWA wordt verder onderzoek ingesteld. Ook de Belgische autoriteiten zijn op de hoogte gebracht van de bevindingen. Op het Nederlandse bedrijf zijn geen dieren meer aanwezig. De AID sluit niet uit dat de komende periode nog meer dieren worden bemonsterd naar aanleiding van analyse van transportbewegingen. bron: AID, 09/10/08

13 OVERVIEW of PRESENTATION 13/36 Biomarkers BioCop Results: identification SPR prototype profiling Take-home messages

14 What is BioCop about 14/36 The fundamental objective of BioCop is to utilise emerging bioscience technologies to create a powerful new approach to detect and control chemical contaminants in foods

15 Change circumstance vs Response 15/36 Practice of Abuse spectrum of agents increases use of masking agents exogenous versus endogenous cocktails relative low concentrations design of new compounds dermal applications metabolism large product volumes Changes in environment may be very subtle and complex; changes may be overlooked easily. Biomarker Response, however, is similar! Classical analysis fails in many ways

16 Project overview 16/36 Hormone treatment studies Biomarker identification in experimentally treated animals 2D-DIGE MS proteomic analysis Conventional assay analysis SPR biosensor based biomarker assay development Biomarker material - Commercial source - Produce recombinant protein Biomarker binder - Commercial source - Antibody production Biomarker screening method based on multichannel biosensor

17 OVERVIEW of PRESENTATION 17/36 Biomarkers BioCop Results: identification SPR prototype profiling Take-home messages

18 Treatment regime 18/36 Veal calve treatment study 10 week old, Holstein Friesian crossbred calves Adult treatment study month old, continental crossbred Steers: E 2 (0.15mg/kg Bwt) Heifers: T (0.85mg/kg Bwt) 17β-Oestradiol benzoate Nortestosterone decanoate Dexamethasone Day Bodyweight gain veal E 2 /NT/Dex - Treated male calves ~7% E 2 /NT/Dex - Treated female calves ~12% adult E 2 -Treated steers ~10% NT-Treated heifers ~5%

19 Biomarkers: identification [1] 19/36 Affinity assays SPR Western-blot ELISA

20 Biomarkers: identification [2] 20/36 Plasma component N-terminal propeptide of type III procollagen (PIIINP) Assay method RIA Treatment E 2 /NT DEX Response in treated females Response in treated males Osteocalcin ELISA DEX Sex-hormone binding globulin (SHBG) Insulin-like growth factor binding protein-3 (IGFBP-3) Urea DHT-binding assay Western blot Clinical analyzer E 2 /NT E 2 /NT E 2 /NT DEX Thyroxine (T4) Clinical analyzer DEX

21 Biomarkers: identification [3] 21/36 2DGE / DIGE image analysis spot picking tryptic digest MS analysis

22 Biomarkers: identification [5] 22/36 2D-DIGE MS proteomic analysis Plasma component E 2 /NT Treatment P-value Alpha-2 antiplasmin precursor Serotransferrin precursor Endopin-1 precursor Plasma component Dex Treatment P-value Serotransferrin precursor Fetuin-A L-lactate dehydrogenase B Alpha-1 antitrypsin precursor 0.019

23 Biomarkers: identification [6] 23/36 Identified candidate plasma proteins (presentation M. Mooney) Biomarker alpha1 antitrypsine precursor E2/NT/DEX not determined alpha2 antiplasmin precursor E2/NT not determined BGLAP(osteocalcin) short after DEX treatment short after DEX treatment Endopin-1 precursor E2/NT not determined IGFBP-2 after second E2+NT treatment after second E2+NT treatment IGFBP-3 E2+NT treatment E2+NT treatment Inhibin asu E 2 +NT treatment unchanged Fetuin-A E 2 +NT treatment not determined How to detect 8 biomarkers simultaneously in a sample? L-lactate dehydrogenase B DEX N-terminal propeptide of type III procollagen E2/NT after DEX unavailable Serotransferrin DEX DEX SHBG - E2+NT treatment Thyroxin (T4) DEX DEX Urea E2+NT treatment after DEX after E2+NT treatment after DEX

24 OVERVIEW of PRESENTATION 24/36 Biomarkers BioCop Results: identification SPR prototype profiling Take-home messages

25 Biomarkers: application 25/36 Surface Plasmon Resonance (SPR) multi-channel biosensor Prototype Biosensor: 16 marker concentrations per sample - 4 running conditions Improved analysis speed: 12 animals / 3 hours Automated data quality control/data evaluation Profile recognition

26 Biomarker Analyzer: flow cells and detection spots 26/36 Out 4 Flow cells In Each Flow cell has 4 detection spots Immobilization spot 100 x 500μm = 0.05mm 2 BC3000: 1.15 mm 2 (23x) Detection spot 80 x 200μm = mm 2 BC3000: 0.27 mm 2 (17x)

27 Experimental Approach [1] 27/36 model buffer -AB response spk in buffer IC50 = sample +AB sample -AB (=NSB) sample -NSB sample log conc. analyte Sensogram: response sample + antibody NSB-correction High response Low [Biomarker] High [Biomarker] Low response time

28 Experimental approach [2] 28/36 Running buffer? Sample pretreatment (heat treatment)? Calibration & Optimization: Determine IC 50 of BM in buffer sensitive range Determine EC 50 of endogenous BM plasma dilution rate vs. sensitive range Response (RU) calibration curve 600 Relresp (NSB corrected) nhi 500 IC 50 = 1690 ng/ml Relresp (NSB corrected) HI IC 50 = 1624 ng/ml Response (RU) alpha2 antiplasmin in adult bovine plasma at various dilution Relresp (NSB corrected) nhi_ap EC 50 = 208x Relresp (NSB corrected) HI_AP EC 50 = 218x log conc. alpha2 antiplasmin buffer (ng/ml) log of plasma dilution rate

29 Running multiple assays 29/36 Chip surface layout for 8 BMs with optimized assays: BM Coupling Method AB IC50 Range in undiluted plasma EC50 dilution rate plasma BGLAP ph4_amino M690 ~ 30 ng/ml ng/ml 0 20 x IGFBP-2 YM10_pH4_amino BC62 ~ 100 ng/ml ng/ml 2 50 x Flow Cell 1 PIIINP (Nt) ph4_ligand thiol BC42 ~ 30 ng/ml ng/ml 2 20 x PIIINP (Ct) ph4_ligand thiol BC47 ~ 80 ng/ml 2-8 µg/ml 2 20 x 2 SHBG YM10_pH4_amino direct assay x x 100x alpha2 antiplasmin alpha 1 antitrypsin YM10_pH4_amino BC57 ~ 1600 ng/ml 80 µg/ml x YM10_pH4_amino BC55 ~ 250 ng/ml 1-3 mg/ml x serotransferrin YM10_pH4_amino BC73 ~ 1000 ng/ml mg/ml x 3 4 fetuin A YM10_pH4_amino anti-fetuin ~ 2000 ng/ml mg/ml 1500 x

30 OVERVIEW of PRESENTATION 30/36 Biomarkers BioCop Results: identification SPR prototype profiling Take-home messages

31 SHBG 31/36 Female veal calves Male veal calves DHT-binding (RU) * * ** ** +++ *** Control Treated DHT-binding (RU) * *** Control Treated Study day Study day E 2 E 2 DEX E 2 E 2 DEX

32 Serotransferrin 32/36 Treated female veal calves Treated male veal calves STF (ug/ml) STF (ug/ml) Treatment Day Treatment Day DEX DEX

33 Other Biomarkers 33/36 Treated male veal calves Treated veal calves Alpha-2 antiplasmin (ug/ml) Alpha-1 antitrypsin (ug/ml) Study day 0 TM35 TM36 TF35 TF36 E 2 E 2 DEX DEX DEX

34 SPR biomarker profiling 34/36 Biomarker Profile Support Vector Machine Treated/Untreated IGFBP-2 Osteocalcin PIIINP Serotransferrin Fetuin-A Alpha-1 antitrypsin Alpha-2 antiplasmin SHBG Day

35 Take Home Messages 35/36 The EC is aware of the cat and mouse game between inspection and farmers/vets and is searching for and invests in alternative detection systems Effect measurements in the animal: new concept of filtering suspected samples HTS screening of biomarker profiles in animal materials (plasma) at the farm, in-/on-/at slaughter-line will assist conventional analysis and improve monitoring systems A very advanced & innovative MA-biosensor is fit for that purpose For more detailed information and news about project events: please visit our website at

36 Acknowledgements 36/36 UU QUB CER Giuseppe Cacciatore Chris Elliott Philippe Delahaut Tesy Merkx Mark Mooney Anne-Catherine Huet Aldert Bergwerff Chen Situ Xiao-Lei Xia Kang Li CART BiaCore Fusion Antibodies Mike Collodoro Jos Buijs Jill Caswell Anne-Cécile Massart Esa Stenberg Philip Snoddy Edwin DePauw Richard Buick Joel Widart

37 Thank you for your attention

38 Running multiple assays 2000 Alpha-1 antitrypsin 500 Alpha-2 antiplasmin Alpha-1 antitrypsin (ug/ml) Alpha-2 antiplasmin (ug/ml) * TM35 TM36 TF35 TF36 Fetuin-A Study day * # # Fetuin-A (ug/ml) [STF] ug/ml TM35 TM36 TF35 TF Day

39 2000 Response (RU) 1000 Thank you for your attention log of dilution rate A B [STF] ug/ml [STF] ug/ml * # # C [STF] ug/ml Day D [STF] ug/ml Day # # Day Day

40 Effects: growth rate DEX 225 E 2 /NT E 2 /NT Relative weight gain (%) E 2 /NT Age (weeks) Control Treatment

41 Biomarkers: identification [6] Biomarker Assay standard Assay binder Fetuin-A Bovine fetuin-a Polyclonal antibody IGFBP-2 Recombinant bovine IGFBP-2 Polyclonal antibody Sex-hormone binding globulin (SHBG) None 1α-aminohexyl-17β-hydroxy- 5α-androstan-3-on N-terminal propeptide of type III procollagen (PIIINP) Synthetic C-terminal PIIINP peptide Synthetic N-terminal PIIINP peptide Polyclonal antibody Polyclonal antibody Alpha-2 antiplasmin Recombinant bovine alpha-2 antiplasmin Polyclonal antibody Alpha-1 antitrypsin Recombinant bovine alpha-1 antitrypsin Polyclonal antibody IGF-1 Human IGF-1 Polyclonal antibody Myostatin Synthetic bovine myostatin ( ) Polyclonal antibody Inhibin Recombinant inhibin (1-32) Polyclonal antibody Osteocalcin Synthetic human osteocalcin (37-49) Polyclonal antibody Serotransferrin Recombinant serotransferrin Polyclonal antibody L-lactate dehydrogenase B-chain Recombinant L-lactate dehydrogenase B chain Polyclonal antibody Endopin-1 Recombinant endopin-1 Polyclonal antibody