Automated Plate ELISA and Dot-Blot Assays ... - Pall Corporation

Scientific & Technical Report PN 33293Automated Plate ELISA and Dot-Blot AssaysUsing AcroWell 96 Filter Plates and a RoboticWorkstation with Integrated Plate ReaderBy Norina M. Tang, Ph.D. 1 and Lois Tack, Ph.D. 21Pall Life Sciences, 600 South Wagner Road, Ann Arbor, MI 48103, USA. Norina_tang@pall.com2PerkinElmer Life Sciences, 2200 Warrenville Road, Downers Grove, IL 60515, USA. Ltack@packardbioscience.comIntroductionThe near completion of the Human GenomeProject, and many other genetic sequencingprojects in recent years, have brought on theproteomics era. Scientists now have the greaterchallenge of identifying all of the resulting geneproducts and assigning functions to therespective proteins. One classical technique forprotein analysis is the western immunoblot that,although highly specific, is time-consuming,technically difficult, and not very sensitive.Moreover, the technique is low-throughputand difficult to automate. In response to thesevarious technical difficulties and limitationsassociated with the western immunoblot,Pall Corporation (East Hills, NY), a leader inmembrane filtration and product design,teamed up with PerkinElmer Life Sciences(Boston, MA, USA), an industry leader in thefield of automation and equipment, to developan alternative assay for the detection ofprotein-protein interactions. The alternativeassay that is described in this study uses96-well membrane-bottom plates to specificallyseparate and capture proteins of interest thatare then detected by a chemiluminescent orfluorescent reader.Historically, the filtration of samples in a 96-wellformat has exposed a variety of problemsincluding well-to-well variation, inability toempty all wells, stacking and handling issues,solution weeping, and high background levels.These issues impact automation affecting bothdata integrity and preventing hands-free, walkawayprocedures. Most problems associatedwith plate filtration are due to the structure andquality of the multi-well filter plates as well as thevacuum manifold design. Through careful designand engineering, Pall Corporation has developedthe AcroWell 96 polypropylene filter platecontaining various high performance membranes.The membranes are sealed to the polypropylenehousing using a patented process thatdramatically reduces crosstalk and weeping.AcroWell 96 filter plates meet Society forBiomolecular Screening (SBS) recommendationsfor automated handling and are ideal for rapidand specific biomolecule detection using roboticsystems. AcroWell 96 filter plates are availablein various colors to help optimize detection.In this study, we demonstrate the use ofAcroWell 96 filter plates with BioTrace membranes for automated plate ELISA anddot-blot applications. We show data for thechemiluminescent and fluorescent detection ofspecific protein binding. We then show data forthe immunodetection of CDK2 in crude humanT-cell lysates. These assays were carried outboth manually and using a fully automatedmulti-instrument system from PerkinElmerLife Sciences consisting of a multi-mode platereader integrated with a liquid handling platform.The specific robotic workstation used consistedof the MultiPROBE* II HT EX workstation withGripper* Integration Platform, Vacuum FiltrationOption, and Fusion* Universal Microplate Analyzer.

Workstation and MaterialsThe assays in this study were performed using aMultiPROBE* II HT EX workstation with Gripper* IntegrationPlatform and Fusion* Universal Analyzer. The PackardVacuum Filtration Option was also included. 1, 2The vacuum filtration system with PVM manifold iscompatible with fully automated processing of Pall’sAcroWell 96 binding filter plates. In this study, vacuumfiltration was carried out at 10 to 12 in. Hg.Pall’s AcroWell 96 filter plates are available in clear, white, andblack housing colors. Choose clear plates for time-resolvedfluorescence, 3 white plates for chemiluminescence/radioactivity, and black plates for fluorescein detectionassays. Plates are also offered in a variety of high-performancemembrane options and device configurations.MultiPROBE II specialized labware was used to automatethe Pall AcroWell 96 filter plate assays for this study.Various colored AcroWell 96 Filter PlatesMultiPROBE II HardwarePackardPart Number DescriptionAMP8E02 MultiPROBE II HT EX/ComputerAMPGRE0 Gripper Integration Platform7401489 ACB100 (100/60)2004082 GAST Vacuum Pump (115/60)7800483/B VersaTip* Plus Software7601811 Vacuum Filtration Option6000656 200 mL Conductive Tips5078889 Tile, Troughs, MPII HT5079598 Trough Holder, 60 mL, 4 Pos.6008104 Troughs, 60 mLA153604 Fusion* HT Universal Analyzer7400139 Fusion: Blue PMT7009577 Fusion Integration Software7801539 Fusion Remote Control SoftwareAcroWell 96 Filter Plate Features• Polypropylene construction for low non-specific binding• High performance membranes (e.g., PVDF, nitrocellulose)• Automation-friendly• No crosstalk between wells• No weeping of solutions• Range of available housing colors• One-piece housing construction prevents jammingin instrumentsAcroWell 96 Filter PlatesPall Part No. DescriptionPN 5027 AcroWell 96 Filter Plate with BioTrace PVDF Membrane, white, 10/pkgPN 5025 AcroWell 96 Filter Plate with BioTrace NTMembrane, black, 10/pkg

Results — ChemiluminescenceProtein Binding Capacity and Detection SensitivityThe first step in the dot-blotting procedure is the binding ofthe target protein to the membrane. For our first experiment,varying amounts (2.5-1000 pg) of HRP-labeled goat IgGwere added to prewetted white AcroWell 96 PVDF filterplates for 30 minutes and washed. LumiGLO* was addedfor 5 minutes. The plates were read automatically using theintegrated Fusion* reader (luminescence mode) or manuallyusing a PerkinElmer MICROBETA* Counter. 4The results indicate that as little as 2.5 pg of goat IgG canbe reliably detected using the AcroWell 96 filter plate systemas described. Moreover, the linear range of binding anddetection is over 3 orders of magnitude.Figure 1MultiPROBE* II Deck Layout and Pall Protocol OutlineAutomated:Figure 2Protein Binding to BioTrace PVDF MembraneBound Protein, RLU (n=11)RLU8000070000R 2 = 0.999660000500004000030000200001000000 200Protein Binding to PVDF MembraneUsing Automated Protocol400 600 800 1000 1200Goat IgG-HRP (pg)6000050000R 2 = 0.9944000030000200001000000 10 20 30 40 50 60Goat IgG-HRP (pg)Protein Binding to PVDF MembraneUsing Manual Protocol140000WinPREP* software automated the assay shown below.Manual:Manual Protocol1. Prewet PVDF membrane2. Wash3. Bind HRP-Antibody4. Wash5. Blot off excess fluid6. Add LumiGLO7. ReadAvg LCPS (n=8)Avg LCPS (n=8)120000R 2 = 0.99381000008000060000400002000000 200400 600 800 1000 1200Goat IgG-HRP (pg)700006000050000R 2 = 0.99294000030000200001000000 10 20 30 40 50 60 70 80Goat IgG-HRP (pg)For ordering or technical information call your local.Pall Life Sciences office listed on the back page.www.pall.com/lab

Specific Protein BindingThe second experiment demonstrates the use of theAcroWell 96 filter plate for the immunodetection of anabundant, purified protein.Varying amounts (1-8 ng) of rabbit IgG (Kirkegaard & PerryLaboratories) were added to prewetted white AcroWell 96filter plates with BioTrace PVDF membrane for 30 minutesand washed. Following incubation with Block (30 minutes)and washing, HRP-labeled goat anti-rabbit IgG was addedfor 60 minutes and washed. Following LumiGLO* additionfor 5 minutes, the plates were read as before. 4Figure 3MultiPROBE* II Deck Layout and Pall Protocol OutlineAutomated:These data show that the signal measured from the secondaryantibody correlated directly to the amount of target proteinpresent. Under these experimental conditions, the specificbinding and detection of the target rabbit IgG proteins islinear from approximately 1 to 10 ng.Figure 4Specific Protein BindingAvg RLU (n=8)100000900008000070000R 2 = 0.9909Specific Protein BindingUsing Automated Protocol60000500000 2 4 6 8 10Rabbit IgG (ng)WinPREP* software automated the assay shown below.Manual:Manual Protocol1. Prewet PVDF membrane2. Wash3. Bind target proteins4. Wash5. Block6. Wash7. Bind HRP-Antibody8. Wash9. Blot plate10. Add LumiGLO11. ReadAvg LCPS (n=3)Specific Protein BindingUsing Manual Protocol120000R 2 = 0.976111000010000090000800007000060000500000 2 4 6 8 10Rabbit IgG (ng)

Detection of CDK2 from Jurkat CellsTo more rigorously test the sensitivity and dynamic range ofthe AcroWell 96 filter plate system, a third experiment wasperformed to specifically bind and detect cyclin-dependentkinase 2 (CDK2) in Jurkat cells. Varying amounts (1-5000 ng)of Jurkat cell lysate (BD Transduction Labs) were added toan AcroWell 96 PVDF filter plate for 30 minutes and washed.After blocking for 30 minutes and washing, anti-CDK2 mAb(BD Transduction Labs) was added for 60 minutes andwashed. HRP-labeled goat anti-mouse IgG was added for60 minutes and washed. LumiGLO* was added and plateswere read after 5 minutes as before. 4Figure 5MultiPROBE* II Deck Layout and Pall Protocol OutlineThese data show that using the AcroWell 96 filter platesystem, CDK2 protein kinase from crude cell lysates canbe specifically detected linearly over one order of magnitude.The exact range of detection will vary depending onexperimental conditions.Figure 6Detection of CDK2 from Jurkat Cells1000095009000Detection of CDK2 from Jurkat CellsUsing Automated ProtocolAutomated:RLU (n=8)8500800075007000650060001 10 100 1000Jurkat Cell Lysate (ng)50000Detection of CDK2 from Jurkat CellsUsing Manual ProtocolWinPREP* software automated the assay shown below.Manual:Manual Protocol1. Prewet PVDFmembrane2. Wash3. Bind cell lysate4. Wash5. Block6. Bind primaryAntibody7. Wash8. Bind HRP-Antibody9. Wash10. Blot excess fluid11. Add LumiGLO12. ReadLCPS (n=2)45000400003500030000250001 10 100 1000 10000Jurkat Cell Lysate (ng)For ordering or technical information call your local.Pall Life Sciences office listed on the back page.www.pall.com/lab

Filter Plate Compared to WesternImmunoblot TechniqueThe results presented above in Figure 6 were comparedto results obtained from traditional western immunoblottechniques for the detection of CDK2 from Jurkat cells.Indicated amounts of Jurkat cell lysate were eletrophoresedand electroblotted to a BioTrace PVDF membrane.Following blocking and washing, the membrane blot wasincubated with anti-CDK2 mAb (60 minutes), washed andincubated with HRP-goat anti-mouse IgG (60 minutes). Afterwashing, the blot was treated with LumiGLO* (5 minutes)and exposed to X-ray film for indicated times.Compared to the AcroWell 96 filter plate technique, thewestern immunoblot technique was 10- to 100-fold lesssensitive (using identical reagents) in detecting CDK2 kinasefrom Jurkat cell lysate. In addition, the AcroWell 96 filter platetechnique required only 3.5 to 4.5 hours to complete whilethe western immunoblot technique required 8 or more hours.Results — FluorescenceProtein Binding Capacity and Detection SensitivityAcroWell filter plates can also be used for fluorescencedetection. Similar to the chemiluminescent results of Figure 2,the results below show that nanogram or sub-nanogramamounts of protein can be detected. In addition, the linearbinding and detection range is over 3 orders of magnitude.Varying amounts (0.5-500 ng) of fluorescein-labeled rabbitanti-goat IgG (Kirkegaard & Perry Laboratories) were addedto a white AcroWell 96 filter plate with BioTrace NTmembrane for 5 minutes, washed and blotted. Plateswere automatically read using the integrated Fusion*reader (top read fluorescence mode).Figure 8MultiPROBE* II Deck Layout for a Fluorescent-labeledProtein Binding ProtocolFigure 7Protein Electro-blotting Technique for theChemiluminescent Detection of CDK2 from Jurkat CellsA. Short exposure (20 seconds)kDa202133714231MW 0.5 1.0 2.5 5.0 .5 10 15µg of Cell LysateB. Long exposure (20 minutes)kDa202133714231MW 0.5 1.0 2.5 5.0 .5 10 15µg of Cell LysateFigure 9Automated Fluorescent Protein Binding140000Automated F-RAG IgG Binding Assay120000Avg RFU (n=8)1000008000060000400002000000 100 200 300 400 500 600Added Protein (ng)

Specific Protein BindingVarying amounts (0.25-50 ng) of goat IgG were added to ablack AcroWell 96 filter plate with BioTrace NT membranefor 30 minutes and washed. Block (BSA, Kirkegaard & PerryLaboratories) was added for 5 minutes and washed.Fluorescein-labeled rabbit anti-goat IgG was added for 60minutes and washed. Plates were read using the VICTOR*1420 Multilabel Counter.The data below demonstrate that the antibody:antigeninteraction detected by fluorescence is highly specific andsensitive. As anticipated, the linear range is 10-fold and, inthis particular case, from 3 to 50 ng of antigen.Figure 10Manual Fluorescent Specific Protein BindingFluorescein CPSManual Protocol1. Bind proteins toNT membrane2. Wash3. Block4. Wash4000035000300002500020000150001000050000Specific Protein Detection UsingFluorescent-labeled Antibody0 0.25 0.5 1 2 3 6 12.5 25 50Goat IgG (ng)5. Bind fluorescein-Antibody6. Wash7. Blot excess fluid8. ReadConclusionsPall AcroWell 96 Filter Plates• The use of the AcroWell 96 membrane-bottom filter plateis an attractive alternative to western immunoblot becauseof its simplicity, rapidity, automation capability, andincreased sensitivity.• Compared to classical western immunoblot, the AcroWell 96filter plate technique can be up to 100-fold more sensitive.• AcroWell 96 filter plate assay takes approximately half thetime or less to complete compared to westernimmunoblot assay.• AcroWell 96 filter plate’s polypropylene housing and lowhold-up configuration using SBS robotic guidelines resultin low non-specific binding, consistent CV’s, greaterdetection accuracy, and automated processing.• The variety of plate housings available for AcroWell 96 filterplates allows optimized detection by chemiluminescent,radiometric, and fluorescent techniques.• The use of high-performance binding membranes such asBioTrace PVDF and NT, permits strong, reproducible bindingresulting in improved specific detection of biomolecules.MultiPROBE* II Analysis Workstation• Workstation features fully automated pipetting gripper tomove plate, and vacuum, timer control, plate read, anddata file storage functions.• Custom software integration package provides interfacebetween WinPREP* script functions and Fusion*instrument control software.• Multi-instrument platform carries out complex protocolsthat include assay set up and selection of desired FusionAssay Definition (e.g., luminescence or fluorescence).• Gripper* Integration Platform provides large off-deck workarea ideal for integrating multi-mode plate readers such asFusion without losing valuable deck space.• WinPREP software supports multipart protocols (e.g., CDK2detection assay protocol contains 17 separate pipettedispense and 17 vacuum steps). Software allows dispensingLumiGLO* substrate in same order as plate read format tominimize time sensitivity of luminescence assays.• Gripper with theta rotational function correctly positionsassay plates in the plate reader carrier.• Precision control of time-sensitive assays using WinPREPtime marker and Fusion software functions fully automatepipetting gripper move plate, vacuum, timer control, plateread, and data file storage functions.For ordering or technical information call your local.Pall Life Sciences office listed on the back page.www.pall.com/lab

References1. Tack, L.C., Holly, D., Galdar, L., and Staat, S. 2001.“Automated Protein and Nucleic Acid Analysis Using aRobotic Liquid Handling Workstation and Multi-ModePlate Reader.” PerkinElmer Liquid Handling ApplicationNote LHA-022.2. Tack, L.C., Van Dinther, J., and Ahrweiler, P. 2001.“Fully Automated Nucleic Acid Extraction Using theMultiPROBE* II HT EX with Gripper Integration Platformand PVM Vacuum Filtration System.” PerkinElmer LiquidHandling Application Note LHA-017.3. Valenzano, K.J., W. Miller, J. Kravitz, P. Samama, D.Fitzpatrick and K. Seeley. 2000. “Development of aFluorescent Ligand-Binding Assay Using the AcroWellFilter Plate.” J. Biomolecular Screening 5(6):455-461.4. Tang, N.M. 2001. “Chemiluminescent Detection forSpecific Protein Binding Using the AcroWell 96 FilterPlate with BioTrace PVDF Membrane.” Pall Life SciencesScientific and Technical Report, PN 33234.Additional Literature1. Seeley, K. 2000. “Biomolecule Binding and BlockingProcedures for AcroWell Filter Plates with BioTrace NTand BioTrace PVDF Membranes.” Pall Life SciencesScientific and Technical Report, PN 33189.2. Tang, N., Wilson, D., Seeley, K. 2002. “AutomatedPurification of Combinational Libraries Using AcroPrep 96Filter Plate with GHP Membrane.” Pall Life SciencesScientific and Technical Report, PN 33245.3. Seeley, K., Niemiec, K. 2000. “The AcroWell Filter PlateMinimizes Crosstalk.” Pall Life Sciences Scientific andTechnical Report, PN 33177.4. “AcroWell 96 Membrane-bottom Filter Plates.” Pall LifeSciences Product Data, PN 33296.Pall LIfe Sciences600 South Wagner RoadAnn Arbor, MI 48103-9019 USA800.521.1520 toll free in USA734.665.0651 phone734.913.6114 faxAustralia – Lane Cove, NSWTel: 02 9428-23331800 635-082 (in Australia)Fax: 02 9428-5610Austria – WienTel: 043-1-49 192-0Fax: 0043-1-49 192-400Canada – OntarioTel: 905-542-0330800-263-5910 (in Canada)Fax: 905-542-0331Canada – QuébecTel: 514-332-7255800-435-6268 (in Canada)Fax: 514-332-0996800-808-6268 (in Canada)China – P. R., BeijingTel: 86-10-8458 4010Fax: 86-10-8458 4001France – St. Germain-en-LayeTel: 01 30 61 39 92Fax: 01 30 61 58 01Lab-FR@pall.comGermany – DreieichTel: 06103-307 333Fax: 06103-307 399Lab-DE@pall.comIndia – MumbaiTel: 91-22-5956050Fax: 91-22-5956051Italy – MilanoTel: 02-47796-1Fax: 02-47796-394or 02-41-22-985Japan – TokyoTel: 3-3495-8319Fax: 3-3495-5397Korea – SeoulTel: 2-569-9161Fax: 2-569-9092Poland – WarszawaTel/Fax: 22-835 83 83Russia – MoscowTel: 095 787-76-14Fax: 095 787-76-15SingaporeTel: (65) 389-6500Fax: (65) 389-6501Spain – MadridTel: 91-657-9876Fax: 91-657-9836Sweden – LundTel: +46 (0)46 158400Fax: +46 (0)46 320781Switzerland – BaselTel: 061-638 39 00Fax: 061-638 39 40Taiwan – TaipeiTel: 2-2545-5991Fax: 2-2545-5990United Kingdom –PortsmouthTel: 023 92 302600Fax: 023 92 302601Lab-UK@pall.comVisit us on the Web at www.pall.com/labE-mail us at Lab@pall.com©2003 Pall Corporation. Pall, , AcroWell, and BioTrace are trademarks of Pall Corporation. The ®indicates a trademark registered in the USA.is a service mark of Pall Corporation.*Packard MultiPROBE II, MICROBETA, and WinPREP are registered trademarks, and Fusion, Gripper,VersaTip, and VICTOR are trademarks of PerkinElmer, Inc. LumiGLO is a registered trademark of Kirkegaard &Perry Laboratories, Inc.02/03, 2.5k, GN02.0624 PN 33293