Proteinmicroarrays - Antikörper im analytischen Einsatz

Transcription

1 Proteinmicroarrays - Antikörper im analytischen Einsatz Dr. Markus Ehrat Zeptosens A Division of Bayer Schweiz AG APPLICA Juni 2009 What are Microarrays? A powerful technology to measure thousands of samples, spots genes or proteins in a short period of time spots Feature size µm Feature size µm 1

2 Entries in PubMed Database, Search Term Microarray all fields protein arrays Bioanalytical Trends - Increase in Information per Sample Detection area (mm 2 ) # of Antibodies per spot* Information per cm 2** 96 well plate 40 4 x well plate x µm spot microarray 1.7 x x µm spot microarray 1.7 x x µm spot microarray 1.7 x x * Assumption: 1 antibody occupies about 100 nm 2 ** Sample solution of 200 µl 2

3 Protein Microarrays Two Formats Forward Protein Arrays Reverse Protein Arrays Array of target-specific capture molecules (e.g. antibodies) Specific detection via labeled secondary Ab (ELISA-type) Two Ab s per target Array of samples Target proteins on chip Specific detection with target-specifc antibodies One Ab per target Forward Protein Assay Formats Signal Sandwich Format Signal Competitive Format Conc. Conc. 3

4 Dose Precision Optimization: Slope and CV of Signal signal 1.2 signal CV 0.8 signal CV k 10k dose CV dose CV (> 20%) dose (< 20%) CV log c (pg/ml) > 20% 28-plex Assay Minimizing Crossreactivities of the Ab Pairs 28 capture (primary) Ab s 28 reporter (secondary) Ab s 28 different analytes, measured in single-analyte assays in the presence all 28 reporter Ab s (sequential assay) False color plot of assay signals generated by all combinations of sandwich duo-pair complexes green = low signal red = high signal In collaboration with GlaxoSmithKline Capture Ab Analyte (+28 reporter Ab s) 4

5 Optimize Assays for Lowest Crossreactivities A B C D E A B C D E Sample Preparation A B C D E A B C D E A B C D E A B C D E Capture Elements A B C D E A B C D E A B C D E A B C D E Assay Conditions A B C D E A B C D E Parallel Cytokine Assay Human IL-1β, IL-2, IL-4, IL-6, IL-12, IL-13, TNFα, IFNγ 5 replicate spots/analyte LOD = 1-10 pg/ml hil-01β hil-04 hil-06 hil-12 hifn-γ htnf-α LOQ = 10 pg/ml in serum (dose CV s less than 20%) analyte (pg/ml) 5

6 small molecule Competitive Assay Format labelled receptor protein High signal in absence of analyte Reduced signal in presence of analyte spot spot Chip General Practice for Assay Development find optimum antibody/detection reagent concentration for desired assayworking range (single analyte) adjust spotting conc. to get signals in the same range for all analytes multiplex assay optimize dose-response curves check crossreactivities high analyte concentration middle analyte concentration low analyte concentration 6

7 Optimization of Reagent Concentrations Find optimum antibody/detection reagent concentration for desired assayworking range (single analyte) 2 α-vit. B12 antibody conc. 2 nm <> 1 shift α-vit. B12 antibody 0.5 nm vitamin B12 [ng/ml] Adapt Dose Response Curve to Needed Conc. Range 1.2 APC-streptavidin α-biotin antibody 1.0 Normalized signal affinity shift factor E biotin (ng/ml) 7

8 Multiplexed Competitive Assay LOD* = 70 pg/ml = 70 ppt (* LOD calculated via blank signal + 3 times its standard deviation) Whole milk was spiked with: -1 ng/ml SEB -1 ng/mlaflatoxin Aflatoxin Concentration [ng/ml] LOD = 0.8 ng/ml = 0.8 ppb (* LOD calculated via blank signal + 3 times its standard deviation) SEB Concentration [ng/ml] Capture Arrays Miniaturization has Positive Effects Equal or superior performance to ELISA - Absolute quantification of analytes down to LOD = 1-10 pg/ml and LOQ = 10 pg/ml - Upscale of parallel assay to approx. 30-plex assay demonstrated - Sensitivity is often limited by affinity of antibody - Limitations in the freedom of assay optimization - All assays have to be performed under the same conditions - Crossreactivities need to be carefully addressed Reproducibility - Dose CV s and inter-array CV s < 20% Automation & throughput - Depending on technology and product unattended measurement of up to 360 arrays - Mircotiter plate formats compatible with robotics 8

9 Protein Reverse Microarrays Array of samples Samples are denatured Monolayer of denatured proteins Sample volume 400 pl Sample concentration is ~ 0.2 mg/ml Spot = Content of ~1 cell Specific detection with target-specifc antibodies Microarrays - Small detection areas Nanoliters of sample volumes: Require high detection sensitivity 9

10 Planar Waveguide Principle - High Sensitivity Fluorescence Microarray Detection free tracer surface confined excitation of bound label CCD camera Advantages of Fluorescence Excitation on PWG Conventional excitation ZeptoREADER - Evanescent excitation sample solution support background problems Confocal excitation: ~ no background Evanescent excitation: Separation of excitation and detection directions Ultimate sensitivity Fast time to result Less sample preparation Direct measurement in blood or serum Focus depth ~ 2µm Depth ~ 100nm 10

11 ZeptoMARK Assay Sensitivity Spiking series of recombinant receptor in 2 mg/ml lysate (avg 3 arrays) Spotted and analyzed at 4 dilutions, duplicate spots Average and std dev of 3 arrays 2 (0) + 3 σ c (rec. receptor) / pm LOD of ~2000 receptor protein molecules per spot Zeptosens Reverse Protein Arrays From Cells to Protein Profiles # 1...# n Cellular systems Drug treatment Cell lysis Spotting Sample ~10 5 cells ~ 1 mg tissue Lysis 50 µl lysis Spotting 400 pl spotting 11

12 Six Arrays per Chip 32 Samples per Array 6 Arrays / Chip Ref Ref Ref Ref Zeptosens Buffer Standards Reference Cell lysate (mg/ml) Reference Parallel Monitoring of Proteins Treatment Raf Ab Erk2 Ab P-Erk2 Ab 6 identical Lysate Arrays P-Akt Ab Akt Ab Bcl-2 Ab Effect From Cell Signaling Technology 12

13 Assay and Readout VI III IV V I I II II Assay Raf Ab P-Akt Ab Akt Ab Bcl-2 Ab Erk2 Ab P-Erk2 Ab Microarray & Image Analysis ZeptoREADER TM ZeptoVIEW TM 8 Erk2 P-Raf (Thr202/Tyr204) 8 6 Erk2 P- Akt (Thr202/Tyr204) 8 6 Erk2 P- Akt (Thr202/Tyr204) Bcl Erk2 P- 2 (Thr202/Tyr204) Erk2 P- (Thr202/Tyr204) P-Erk2 (Thr202/Tyr204) 6 0 C T 1T 2 C T 1T C TC 4T C T C T C T C T T B 2 6 Cell line 1 0 C 2 T buffer 1T 2 C T 1T C TC 4T C T C T C T C T T B 2 Cell line buffer C T 1T 2 C T 1T C TC 4 T C T C T C T C T T B 2 Cell line buffer C T 1T 2 C T 1T C TC T C T C T C T C T T B 2 Cell line buffer MEN2A GIST882 Ba/F3 FLT3-ITD MEN2A Ba/F3 bcr-abl KB31 Ba/F3 FLT3-ITD GIST882 A31 (PDGFR) Ba/F3 bcr-abl A431 (EGFR) MEN2A KB31 NWT-21 (IGF-1R) A31 (PDGFR) Ba/F3 FLT3-ITD GIST882 U87-MG A431 (EGFR) Ba/F3 bcr-abl Spotting Buffer MEN2A NWT-21 KB31 (IGF-1R) A31 (PDGFR) U87-MG GIST882 Ba/F3 FLT3-ITD Spotting Buffer Ba/F3 bcr-abl A431 (EGFR) MEN2A KB31 NWT-21 (IGF-1R) Ba/F3 FLT3-ITD A31 (PDGFR) U87-MG GIST882 Ba/F3 bcr-abl A431 (EGFR) Spotting Buffer MEN2A KB31 NWT-21 (IGF-1R) A31 (PDGFR) Ba/F3 FLT3 -ITD U87-MG A431 (EGFR) Spotting Buffer Ba/F3 bcr-abl NWT-21 KB31 (IGF-1R) U87-MG A31 (PDGFR) Spotting Buffer A431 (EGFR) NWT-21 (IGF-1R) U87-MG GIST882 C T 1T 2 C T 1T 2 C TC T C T C T C T C T T Cell line buffer C T1T2 C T1T2 C TC T C T C T C T C C T T B Cell line buffer Protein expression / activation profiles B Spotting Buffer Signatures Guide Drug Choice Julian Downward. Cancer drugs are increasingly designed to target specific cell-signalling pathways. Pathways can be activated at different points [ ]. If a factor at the top of a signalling cascade is unaffected, for instance, one cannot assume that the pathway is not involved, as a factor further downstream might have been activated Nature 439, (19 January 2006) 13

14 Requirements for Pathway Proteomics Approaches Signaling is highly dynamic protein phosphorylations in seconds/minutes protein synthesis/degradation in hours/days extensive sampling required to obtain time resolution Parallel measurements of analytes is important a thorough pathway profile can easily comprise 100 or more elements An array-based solution will provide the required scalability and throughput for extensive mapping of pathway markers and sampling at different time points to capture the cellular signaling events Stimulation with Insulin A431, starvation over night Stimulation with Insulin (150nM) 7 time points, min 0.3 P-Akt (S473) Insulin InsR IGFR IRSs PI3K (p110,p85) P-S6 Ribosomal Protein (S ) Ctrl EGF(t) W(t) W+EGF(t) W(c)+Ins Ctrl EGF(t) Ins(t) W(t) W+EGF(t) Cell growth W(c)+Ins mtor S6 kinase S6 Ins(t) PIP 3 PIP 2 PTEN PDKs PKB/Akt GSK3 Glycogen synthase Ctrl EGF(t) P-GSK-3b (Ser9) W(t) W+EGF(t) W(c)+Ins Ins(t) 14

15 Inhibition with Wortmannin A431, starvation over night Treatment with Wortmannin, 1h inhibitor conc., 0-3µM 0.3 Stimulation with Insulin, 1h Ctrl EGF(t) W(t) W+EGF(t) 0 0 W(c)+Ins 0.1 Ctrl EGF(t) P-S6 Ribosomal Protein (S ) Ins(t) P-Akt (S473) W(t) W+EGF(t) Cell growth W(c)+Ins mtor S6 kinase S6 Ins(t) PKB/Akt GSK3 InsR IGFR IRSs PI3K (p110,p85) PIP 3 PIP 2 PTEN PDKs Glycogen synthase Insulin Ctrl EGF(t) W(t) W+EGF(t) Wortmannin P-GSK-3b (Ser9) W(c)+Ins Ins(t) Compound Profiling using Reverse Protein Arrays P-Akt (Ser473) IGF receptor trec 50 = 171 nm PTEN PI3K (p110,p85) IRSs inhibitor % Inhibition PIP 2 PIP 3 PDKs 20 0 ctrl Log Concentration (nm) FHs (AFX1,FKHR) Transcription PKB/Akt GSK3 Glut4 Glucose transport Regulation of glycogen synthase % Inhibition P-GSK3beta trec 50 = 263 nm ctrl Log Concentration (nm) trec50" (transduced EC50) curves of an IGFR inhibitor at different pathway nodes 15

16 Summary of Reverse Protein Array Technology Robust and well-established process Time course of stimulants on activation and expression of pathway proteins Flexible application: free selection of protein profiling targets Straight forward assay development Economic technology - minimal amount of samples and antibodies (<1µL for 2 arrays) needed - short time to result: information corresponding to Western blot lanes within 3 weeks Sensitive Results corroborated by well established technologies as e.g. WB, ELISA Microarrays to Achieve a Clear Overview of Complex Systems Thank you for your attention 16