Alpha Technology. From ELISA to Epigenetics, a FASTER, EASIER way to run complex Assays. Maxine Santoro Ph. D. Field Application Scientist

Transcription

1 Alpha Technology From ELISA to Epigenetics, a FASTER, EASIER way to run complex Assays Maxine Santoro Ph. D. Field Application Scientist 211 Perkin Elmer University of Iowa, October 3, 212

2 Agenda Alpha Technology- How it works/ why it is different Assay Examples- What kind of assays you can run ELISA- Comparison of ELISA vs. AlphaLISA assays Phosphoprotein Detection- Comparison of Western Blot vs. Alpha SureFire Protein:Protein Interactions- Kinase: protein interaction assays Epigenetics Assays How will Alpha Technology help your lab?

3 Principle of the Alpha Technology Bead based assay- Assay reagents link to beads and bring beads into close proximity allowing a signal to be generated Time resolved/low background Singlet oxygen can travel 2nm before decay Low energy to high energy Large amplification due to chemical reaction in Acceptor beads

4 AlphaScreen Technology Summary Amplified Luminescence Proximity Homogeneous Assay Background: Developed by Dade Behring, Inc., in 1994 as LOCI PerkinElmer has rights to technology for research only Latex Bead-based System Donor & Acceptor beads ~25 nm diameter, each with unique chemistries Stable Colloidal Suspension stay suspended (>12 hrs) will not clog pipettes can be centrifuged Coated with a Dextran Polymer Hydrogel prevents non-specific interaction & aggregation contains reactive aldehyde groups for conjugation

5 Biological Interactions Measured with Alpha Technology

6 Biological Interactions Measured with Alpha Technology

7 AlphaLISA immunoassays: assay formats Sandwich Assay Signal Streptavidin-coated Donor bead with biotinylated antibody Competition Assay Analyte Antibody coupled to Acceptor bead More than 1 Ab to Analyte Endogenous analyte Biotinylated Analyte Streptavidin-coated Donor bead with biotinylated antibody Antibody coupled to Acceptor bead Signal Endogenous analyte Competing analyte Few Abs to Analyte or Small Analyte So why do alpha?

8 AlphaLISA comparison to ELISA AlphaLISA ELISA AlphaLISA compare to traditional ELISA? AlphaLISA is bead based, technically different but similar in functionality (sandwich assay in solution) No wash vs wash (time consuming) Scalable (from 96-well to 1536-well) Very wide dynamic range When to consider utilizing AlphaLISA? Targeted analyte (kits) Desire for a simple protocol with no dilutions and wash steps Working with low affinity interactions that can be disrupted by washing Automation, miniaturization & higher throughput needed Easier protocol

9 ELISA vs AlphaLISA ELISA (4-8 hours) Add assay buffer, matrix solution, standard (or sample); Add antibody detection solution Incubate 1 hour shaking then wash well Add enzyme Incubate 3 minutes shaking then wash well 6 3 Add substrate Incubate 3 minutes shaking Read AlphaLISA (2 hours) Add assay buffer, matrix solution, standard (or sample); Add biotin antibody and AlphaLISA acceptor beads Incubate 3-6 minutes (Room Temperature) Add donor beads Incubate 3-6 minutes (Room Temperature) Read

10 AlphaLISA versus ELISA IL8 and IL1β (UMass Medical Center, US) Cell culture supernatants IL- 8 IL 1-Beta 1, 4 8 r 2 = r 2 =.997 ELISA 6 4 ELISA AlphaLISA AlphaLISA very good correlation between the two assays

11 AlphaLISA versus ELISA Insulin kit (Roche, New Jersey) 18 plasma samples tested (diluted 1 times in AlphaLISA buffer) 4 Insulin data correlation between Mercordia ELISA and AlphaLISA kit AlphaLISA insulin (ng/ml) 3 2 R 2 = Mercodia insulin (ng/ml) very good correlation between the two assays

12 Summary of Alpha Benefits Poulsen and Jensen: JBS 12(2) , 27 * LOCI = AlphaLISA > 1 fold > 2 fold > 5 fold Page 12

13 AlphaLISA Effect of serum dilution A human serum sample spiked with 1 µiu/ml was diluted in matrix solution (serum with insulin removed) Linearity (insulin content in uiu/ml) Experiment [expected Dilution insulin] n=1 n=2 n=3 Average % of expected / / / n/a Conclusion: AlphaLISA samples can be diluted with serum (analyte depleted) and the results correspond to the expected dilution a 1:2 dilution yields half the signal Last 4 slides show ELISA and AlphaLISA give expected results-- both good technologies What makes Alpha better than ELISA assays? What makes Alpha better than ELISA assays? )

14 AlphaLISA Assay Precision Assay precision-5 samples spiked with various concentrations of insulin 6 replicates Intra-assay variation (insulin content in uiu/ml) -same plate replicate [spiked insulin] Average %CV Inter-assay variation (insulin content in uiu/ml) -different experiments/days Experiment [spiked insulin] n=1 n=2 n=3 n=4 n=5 n=6 Average %CV Conclusion: Small variation higher concentrations, At lower concentrations nearing LDL (1.5 uiu) variation increases

15 Typical standard curve analysis Dynamic Maximum signal Minimum signal AlphaLISA Signal (counts) 1,, 1, 1, 1, 1 LDL : 1.9 pg/ml - range Log [rat C-Peptide-1] (g/ml) HDL Average (n=12) - 3SD LDL Average bkg (n=12) + 3SD Lower Detection Limit- lowest reading where obtain significant difference from background Dynamic range- Range from lowest detection limit to highest level of detection S/B = maximum signal i i i l

16 Standard Curves of TNF ELISA vs. AlphaLISA Comparison of sensitivity and range of detection 1 4 AlphaLISA AlphaScreen (Counts) ELISA ELISA (OD) [TNF- ] (pg/ml) Lower limit of detection Dynamic Range 15 pg/ml ELISA vs. 1.5 pg/ml AlphaLISA 2 logs for ELISA vs. 4 logs for AlphaLISA

17 mil6 assay on collection of Samples -Standard Curve

18 M IL-6 Graph on Enspire

19 IL6 Alpha Calculated in 5 ul Assay sample number calc values Enspire data Not visible in ELISA

20 5 ul Standard Curve Bottom of curve=68 R2=.97 Goes to.93?

21 Standard Curve for 1 ul Assay LDL=.56 pg/ml Bottom of curve=295 R2=.92 LDL goes to.439

22 Summary of IL6 AlphaLISA -Assays are very easy to do -Very low data variability -commensurate with pipetting s -Very sensitive from 1.2 pg/ml detection -Need to make certain that matrix for sample is the same as for samples -Easy to miniaturize

23 EPO Determination In AlphaLISA buffer: LDL: 1. miu/ml Dynamic range: 1 3, pg/ml In Analyte-depleted serum: LDL: 5.8 miu/ml Dynamic range: 5.8 3, pg/ml

24

25 AlphaLISA Summary AlphaLISA is: EASY: all ELISA assays can be easily converted to the AlphaLISA platform FLEXIBLE: AlphaLISA can detect a broad range of analytes in complex biological matrices KEY advantages of AlphaLISA over ELISA are: Homogeneous (no wash steps) Highly sensitive with a Wide Dynamic Range Simple (3 addition steps) and fast protocol Scalable (96, 384 and 1536 formats) and easily automatable Validated on PerkinElmer Instruments Less hands on time Cost-effective

26 What Else Can you do with Alpha? AlphaLISA Significantly less steps, less time (< 2 hours) Superior dynamic range, great sensitivity Low sample volume- 5 l SureFire PhosphoProtein Detection Comparable results to your Western-Easier Elimination of imaging/ problems with uneven blots Protein: Protein Interaction Assays Ability to use large proteins- unlike Fluorescent polarization or FRET Ability to measure low affinity mm interactions Epigentics Assays Alpha and TR-FRET assays Methylation and Acetylation assays AlphaLISA vs. SureFire Differences

27 Immunoassays ELISA ASSAY CONFIGURATIONS 615 nm AlphaLISA Configuration Immunoassay ELISA assay --Streptavidin Donor beads --Biotin Ab --analyte --Ab Direct conjugation to Acceptor beads nm SureFire Configuration Immunoassay ELISA assay --Streptavidin Donor beads --biotin AB-- --analyte-- --Ab linked to Protein A Acceptor beads Biotinylated Ab can not bind to Protein A Use biotinylated mouse IgG 1 antibody to avoid interference with Protein A.

28 Current AlphaScreen SureFire Kits

29 AlphaScreen SureFire Cellular Kinase Assays General Protocol cell culture Add cells Add Inhibitors Add Stimulant Lyse cells Adherent cells: Incubate overnight Non-adherent cells: Assay immediately Adherent cells: Remove media first or Add concentrated stock direct to wells Non-adherent cells: Add concentrated stock direct to wells Adherent cells: Add concentrated stock direct to wells Non-adherent cells: Add concentrated stock direct to wells Adherent cells: Remove media first and lyse with 1X Lysis buffer or Add 5X Lysis buffer direct to wells Non-adherent cells: Add 5X Lysis buffer direct to wells

30 AlphaScreen SureFire Cellular Kinase Assays General Protocol Transfer lysate from the culture plate to a fresh microplate for assay Perform assay in the culture plate lysate Assay Reagents Assay Target 1 reagents Assay Reagents OR Assay Target 2 reagents Culture plate Culture plate Assay Target 3 reagents Assay plate «Transfer (2 plates) Assay» «Single Plate Assay» «One plate Assay» «One-Well Assay»

31 SureFire vs. Western Blot Detection ERK Standard Curve Recombinant ERK1 Standard Curve Western SureFire Conclusion: Alpha SureFire produces equivalent results to Western Blot

32 SureFire measures ERK-1 activation by agonist more accurately than Western Blot Human Muscarinic Receptor2/ Agonists Conclusion: Alpha SureFire produces equivalent results to Western Blot

33 GPCR Signaling to ERK AlphaScreen Surefire perk assay for GPCR activation Data indicates that most GPCRs can signal thru perk generic assay Large assay windows & S/B

34 Insulin receptor signaling pathway screen for PI3K / AKT / mtor L L P P P PI3K PDK1 AKT mtor P389 p7 S6K Ras Raf MEK ERK P P421/424 P GTP P p7 S6K Phosphorylation (counts) 2 Rapamycin (mtor) 15 Control insulin alone U126 (MEK) Ly2942 (PI3K) Log [Insulin] (M) P7 S6K Phosphorylation (Thr389) in MCF-7 Cells AlphaScreen SureFire Growth & Differentiation Targets

35 SureFire Selectivity Assay Example LY compound inhibits AKT pathway but not MEK pathway. Conclusion: Able to run simultaneous assays using SureFire on one lysate Assay for PI3-kinase i hibit d ifi it Kits

36 Comparison of Alpha SureFire and Western Blot Assays Westerns SureFire Sensitivity High High/Ultra kit Specificity 2 antibodies 2 antibodies Quantification Assay Nature Multiple Targets Relative -based on band intensity Throughput 1-2 lanes per gel Up to unlimited samples in parallel 2 steps 4-5 hrs. hands-on 8-24 hrs total Limited by gel lanes CV s < 5% Normalize with total AKT or GAPDH kits 7 steps hrs hands-on 4-6 hours total Analysis of 5 targets in same pathway from one sample

37 Anything Else you can do? AlphaLISA Significantly less steps, less time (< 2 hours) Superior dynamic range, great sensitivity Low sample volume- 5 l SureFire PhosphoProtein Detection Significantly less steps, less time Elimination of imaging/ problems with uneven blots Protein: Protein Interaction Assays Ability to use large proteins- unlike Fluorescent polarization or FRET Ability to measure low mm affinity interactions Epigenetics Assays Alpha and TR-FRET assays Methylation and Acetylation assays

38 AlphaLISA Toolbox Alpha Donor Bead AlphaLISA Acceptor Bead. Anti-Chicken IgY Anti-cMyc Anti-Digoxigenin Anti-FITC Anti-FLAG Anti-GFP Anti-Goat IgG Anti-GST Anti-His Anti-Human IgG Anti-MBP Anti-Mouse IgG Anti-Mouse IgM Anti-Rabbit IgG Anti-Rat IgG Anti-Sheep IgG Anti-V5 Glutathione Ni chelate Protein A Protein G Protein L Strep-Tactin Streptavidin Protein A Anti-FLAG Anti-Mouse Anti-Rabbit Strep -Tactin Ni chelate GSH Streptavidin You can use this pair This pair might give high background Not recommended

39 Protein:Protein Interaction Assay Examples P53-HDM2 Protein:Protein Assay using tagged proteins --biotinylated P53 protein --GST tagged HDM2 protein --anti-gst Acceptor Bead --Streptavidin Donor Bead- Tag detection Acceptor beads :

40 MEK1-ERK2 kinase activation model Assay background: In the unactive state, unphosphorylated MEK1 and ERK2 are tightly bound. Activated by phosphorylation, MEK1 phosphorylates and activates ERK2, resulting in ERK2 activation and complex dissociation. Using Alpha -Ability to develop an assay measuring protein interaction and dissociation ERK- Kinase MW = 42kD MEK- Kinase MW = 5kD Mathieu Arcand SBS Meeting Biochemistry, Vol. 49, No. 15, 21

41 MEK1-ERK2 activation model ERK1/2 MAP kinase pathway In the unactive state, unphosphorylated MEK1 and ERK2 are tightly bound. Activated by phosphorylation, MEK1 phosphorylates and activates ERK2. This results in MEK1-ERK2 complex dissociation. 41

42 MEK1 ERK2 interaction assay Emission recorded at nm 1 O 2 Excitation 68 nm Ni chelate conjugated AlphaLISA Acceptor Beads Gutathione-coated Donor Beads [MEK1] 1 nm 3 nm 1 nm 3 nm 1 nm MEK1-ERK2 interactions can be detected with an antibody-free AlphaLISA setup, and activation state of either protein greatly influences binding. AlphaLISA Signal (counts) 25, 2, 15, 1, 5, unactive unactive [ERK2 unactive] (M) active unactive [ERK2 unactive] (M) active active [ERK2 active] (M) 42

43 MEK1 kinase assay on ERK2 Anti-pTpY ERK1/2 antibody Emission recorded at nm Excitation 68 nm 1 O 2 Anti-mouse conjugated AlphaScreen Acceptor Beads Gutathione-coated Donor Beads AlphaScreen Signal (counts) 2, 15, 1, 5, [ERK2] (M) [MEK1] 1 nm 3 nm 1 nm 3 nm 1 nm His-tagged active MEK1 was incubated with unphosphorylated GST-ERK2 in the presence of 1 µm ATP 43

44 MEK1-ERK2 phosphorylation-interaction assay Emission recorded at 615 nm Excitation 68 nm 1 O 2 Gutathione-coated Donor Beads Ni chelate conjugated AlphaLISA Acceptor Beads 1 O 2 Anti-pTpY ERK1/2 antibody Anti-mouse conjugated AlphaScreen Acceptor Beads Emission recorded at 572 nm Active His-tagged MEK1 was incubated with unphosphorylated GST-ERK2 in the presence of increasing ATP concentrations AlphaLISA (615 nm counts) 5, 4, 3, 2, 1, [ATP] (M) 17,5 15, 12,5 1, 7,5 5, 2,5 AlphaScreen (572 nm counts) We here provide the first direct evidence that ERK2 phosphorylation triggers its dissociation from active MEK1. Both biomolecular events are intrinsically linked with interaction IC 5 matching ERK2 phosphorylation EC 5. 44

45 Mechanism of Action of Kinase inhibitors Phosphorylation Interaction unactive unactive active unactive No ATP Tightly bound +ATP Loss of binding ATP site competitor Biphasic reduction in interaction assay Due to different binding affinities of Compounds to MEK and ERK Staurosporine AlphaLISA (615 nm counts) 175, 15, 125, 1, 75, 5, 25, IC 5 =.3 nm IC 5 = 15.7 µm Inhibitor [ ] No ATP/ no phosphorylation , 2, 1, EC 5 = 25. nm IC 5 = 129 nm , 15, 1, 5, AlphaScreen (572 nm counts) Loss of phosphorylation activity (staurosporine) Rescues binding at higher concentrations Allosteric inhibitor U126 AlphaLISA (615 nm counts) 175, 15, 125, 1, 75, 5, 25, IC 5 = 1.6 nm IC 5 = 1.4 µm , 2, 1, No ATP/ no phosphorylation Biphasic reduction in interaction assay Conclusion: Different inhibitors display different profiles EC 5 = 15.6 µm IC 5 =.4 nm , 15, 1, 5, Binding activity not rescued By allosteric inhibitor AlphaScreen (572 nm counts)

46 More uses. AlphaLISA Significantly less steps, less time (< 2 hours) Superior dynamic range, great sensitivity Low sample volume- 5 l SureFire PhosphoProtein Detection Significantly less steps, less time Elimination of imaging/ problems with uneven blots Protein: Protein Interaction Assays Ability to use large proteins- unlike Fluorescent polarization or FRET Ability to measure low mm affinity interactions Epigenetics Assays Alpha and TR-FRET assays Methylation and Acetylation assays

47 Epigenetics: The study of non-dna sequence-related heredity This is what determines what genes are expressed during development and involved in cell differentiation and determines if a cells becomes a nerve cell, bone cell etc. #1 #2

48 The Nucleosome Highest profile modifications Writers: Acetylation (HAT) Methylation (HKMT and HRMT) Ubiquitination and Ubl (E1, E2, and E3) Phosphorylation (kinase) Erasers : Deacetylation (HDACs and Sirtuins) Demethylation (demethylase) Readers: Proteins that bind to the modified histones and act H3 H2A H2B H4 Richard Wheeler Our current products are focused on the WRITERS and ERASERs of Histone H3 and p53 with a tool box that allows the development of READER assays

49 In Vitro Enzymatic Assays In vitro Toolbox Toolbox for detecting Histone H3 & p53 modifications Over 3 enzyme assays developed (including new assays for unmodified H3K9/K27 and H3R2me)

50 TR-FRET (LANCE) and AlphaLISA Detection Assays Set-up Methylation and Acetylation assays AlphaLISA set up Excitation 68 nm Emission 615nm Methylate or acetylate biotinylated peptide bio Streptavidin-coated Alpha Donor Bead Modified peptide Anti-mark AlphaLISA Acceptor Bead epigenetic mark TR-FRET set up Same assay using donor and acceptor pairs Excitation 32 or 34 nm FRET TR-FRET Emission 665 nm Residual fluorescent emission 615 nm Eu-labeled antibody epigenetic mark biotin-peptide bio SA-ULight TM Streptavidin-ULight TM LANCE Ultra: Donor : W124 - Eu chelate (conjugated to Ab) Acceptor : ULight (conjugated to Streptavidin)

51 Typical Signal-increase Assay: AlphaLISA EZH2 Methylation Assay 15 ng/well EZH2 complex 1 nm biotin-h3k27me peptide (H ) ATKAARKSAPATGGVKKPHRYRPGGK(B iotin)-oh 3 µm SAM (K m,app ) 12 min reaction at 23 C (linearity verified) Detection with anti-h3k27me2-1 Acceptor beads <1% substrate turnover!! AlphaLISA Signal (counts) 7, 6, 5, 4, 3, 2, * 1, Time (min) [EZH2] (ng/well) [SAM]: 1 µm AlphaLISA Signal (counts) 5, 4, 3, 2, 1, K m app = 2.9 µm Log [SAM] (M) AlphaLISA Signal (counts) 35, 3, 25, 2, 15, 1, 5, IC 5 = 42 M Log [Sinefungin] (M) AlphaLISA Signal (counts) 4, 3, 2, 1, No inhibitor 1 mm Sinefungin Well # Z' =.71 S/B = 52 Optimized assay conditions:

52 AlphaLISA assay set-up with full-length histone H3 Substrate: recombinant Histone H3 (Active Motif) Uses anti-h3k4me1-2 Acceptor beads and biotinylated anti-histone H3 (C-ter) antibody High salt treatment required after the enzymatic assay Unique detection buffer optimized for bead dilution (different from Epigenetics Buffer 1) Histone H3 protein substrate Alpha AlphaLISA Signal (counts) 2, 15, 1, 5, AlphaLISA Signal (counts) 2, 15, 1, 5, # wells Log [Inhibitor] (M) Total 1 mm Sinefungin Z' =.68 S/B = 25 1 mm Sinefungin Z' =.7 S/B = 72 Sinefungin IC 5 = 47 µm SAH IC 5 = 175 µm Validation of SET7/9 assay

53 Epigenetics Portfolio Overview: In Vitro Assays Histone Mark In vitro Detection Cell-based Histone H3 Detection Reagents H3R2me2 H3Thr3p H3K4 H3K4me1-2 H3K4me2 H3K9 H3K9ac H3K9me2 H3Ser1p H3K27ac H3K27me2 H3K27me3 H3K36me2 LANCE & AlphaLISA Tool box for developing in vitro enzyme assays LabChip enzyme kinetics Not covered in this presentation, but also available from PerkinElmer: Radiochemical bio-ab H3 (C-ter) Detection buffer Epigenetics buffer 1 Buffer Set Cell-Histone (3) techniques In Vitro: Cellular: Kits for detection of endogenous histone H3 epigenetic

54 Protocol Summary Preparation Overnight Seed cells in white opaque CulturPlate -384 microplate Treat cells if desired 4 h (adherent cells) overnight Addition Assay volume volume 1 µl 1 µl 5 µl 15 µl Histone Extraction 25 min Cell-Histone Lysis buffer 15 min Cell-Histone Extraction buffer 1 min 5 µl 2 µl 1 µl 3 µl Detection 9 min Acceptor beads (2 µg/ml final) + biotinylated anti-h3 (3 nm final) 6 min Donor beads (2 µg/ml final) 3 min Read plate on EnVision or EnSpire 1 µl 4 µl 1 µl 5 µl 5 µl

55 Cell Titration 5 untreated cells/well 1 NaB-treated cells/well 1 5 1, 2, 5, 1, 1,, 8, 6, 4, 2, untreated 2mM NaB AlphaLISA Signal (counts) 1 5 1, 2, 5, 1, AlphaLISA Signal (counts) 1,, 8, 6, 4, 2, 1 5 1, 2, 5, AlphaLISA Signal (counts) 1, 2, HeLa HEK 293 Jurkat Cells per well Cells per well Cells per well 3, 2, 1, 1 5 1, 2, 5, 1, AlphaLISA Signal (counts) 12, 1, 8, 6, 4, 2, OCI-LY-19 SU-DHL-6 Cells per well 12, 1, 8, 6, 4, 2, AlphaLISA Signal (counts) 1 5 1, 2, 5, 1, Cells per well

56 Selecting the Right Cell Line AlphaLISA Signal (counts) 8, 6, 4, 2, untreated 2 mm NaB HeLa HEK-293 Jurkat Mol. Weight (kda) NaB HeLa HEK-293 Jurkat H3K9ac Total H3 Different cell lines exhibit different mark levels different NaB-fold stimulation Corroboration of Alpha by Western blot data

57 Wigle, SBS 21 (PKI focus group)

58 igle, SBS 21 (PKI focus group)

59 Wigle, SBS 21 (PKI focus group)

60 Wigle, SBS 21 (PKI focus group)

61 Wigle, SBS 21 (PKI focus group)

62 Summary AlphaLISA Assays Significantly less steps, less time Superior dynamic range, great sensitivity Low sample volume- 5 l SureFireAssays- PhosphoProtein Detection Significantly less steps, less time vs. Westerns Elimination of imaging/ problems with uneven blots Protein: Protein Interaction Assays Ability to use large proteins- unlike Fluorescent polarization or FRET Ability to measure low affinity (mm affinities) Epigentics Assays Alpha and TR-FRET assays Methylation and Acetylation assays

63 PerkinElmer Contacts Cathy Sweeny Reagent Technical Specialist Phone: (636) Steven Anderson Instrumentation Sales Specialist Phone: (63) Maxine Santoro Field application Scientist Phone: (734)

64 EnSpire AlphaPLUS AlphaLISA/AlphaScreen detection capability Photometric UV/VIS technology for ELISA assays and DNA/protein quantitation ELISA reference wavelength correction Data export in Excel /text formats to the network or into a USB memory stick Integrated data analysis software: curve fit (lin reg, spline, 4PL/5PL), background subtraction, ratio calculation,ic5 calculation, Average, CV %, and Z value Easy access to the filter wheel with eight barcode identified filter positions Touch Screen: Easy to use; saves desktop space Pre coded assay protocols Up to 384 well plates 21 CFR Part 11 support Windows Vista Operating System