ELISA Technical Guide

Corporate Headquarters IDEXX Laboratories, Inc.One IDEXX Drive, Westbrook, Maine 04092 USATel: 1-207-556-4890 or 1-800-548-9997Fax: 1-207-556-4826 or 1-800-328-5461

European Headquarters IDEXX Europe B.V. Koolhovenlaan 20, 1119 NE Schiphol-Rijk The NetherlandsTel: 31-20-655-23-00 or 00-800-727-43399Fax: 31-20-655-23-33

HerdChek, FlockChek and xChek are trademarks or registered trademarks of IDEXX Laboratories, Inc. in the United States and/or other countries.

© 2007 IDEXX Laboratories, Inc. All rights reserved. • 09-65846-00 (2)i d e x x . c o m

Introduction 1

ELISATechnology 2

ELISAKits 3

ELISAEquipment 5

EquipmentMaintenanceandCalibration 6

ReagentHandlingandPreparation 7

HandlingandPreparingKitComponents 8

QualityControl 9

SampleHandling 10

PipettingMethods 12

ELISAPlateTiming 15

ELISAPlateWashing 16

ReadingPlatesandDataManagement 18

ELISATroubleshooting 19

Appendixes

A:GravimetricPipetteCalibrationProcedure 23

B:FlockChek*andHerdChek*InventoryControlTrackingChart 24

C:LaboratoryTrackingChart 25

D:MaintenanceandCalibrationSchedule 26

E:QualityControlQuickCheck 27

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ELISAIDEXXLaboratoriesmanufacturesdiagnostictestkitsforthedetectionofdiseasesinruminants,horses,swineandpoultry.

Theenzyme-linkedimmunosorbentassay(ELISA)isoneofthemostsensitiveandreproducibletechnologiesavailable.Theseassaysarerapid,simpletoperformandeasilyautomated.IDEXXintroducedthefirstcommercialpoultryELISAforinfectiousbursaldisease(IBD)in1985andthefirstcommerciallivestockELISAforAujeszky’sdisease/pseudorabiesin1986,enhancingthewaylaboratoriestestproductionanimalstoday.

Aswithanyassay,thereproducibilityandreliabilityofELISAsaredependentuponpropertechniqueandattentiontodetail.ThisELISAtechnicalguidewillincreaseyourawarenessofELISAtechniquesandhelpyoumaintainproficiencywiththismethodology.

Checkyourpackageinsertforspecificinstructionsforeachassayyouperform.Periodically,improvementsandrevisionsaremadetokitandpackageinserts.Therefore,itisimportanttoreviewtheprotocolonaregularbasis.Ifyouhavequestionsconcerninganyofthefollowinginformation,callIDEXXTechnicalServicesforassistance.WithintheUnitedStates,call1-800-548-9997or1-800-943-3999.OutsideoftheUnitedStates,call1-207-556-0895andselecttheProductionAnimalServicesTechnicalSupportoption.OrvisitourWebsiteatwww.idexx.com/production/contact/contactpas.jsp.

Introduction

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ELISATheELISAisarapidtestusedfordetectingandquantifyingantibodiesorantigensagainstviruses,bacteriaandothermaterials.Thismethodcanbeusedtodetectmanyinfectiousagentsaffectingpoultryandlivestock.

InELISAtechnology,thesolidphaseconsistsofa96-wellpolystyreneplate,althoughothermaterialscanbeused.Thefunctionofthesolidphaseistoimmobilizeeitherantigensorantibodiesinthesample,astheybindtothesolidphase.Afterincubation,theplatesarewashedtoremoveanyunboundmaterial.Conjugateisthenaddedtotheplateandallowedtoincubate.

Theconjugateconsistsofeitheranantigenorantibodythathasbeenlabeledwithanenzyme.Dependingupontheassayformat,theimmunologicallyreactiveportionoftheconjugatebindswitheitherthesolidphaseorthesample.Theenzymeportionoftheconjugateenablesdetection.

Theplatesarewashedagainandanenzymesubstrate(hydrogenperoxideandachromogen)isaddedandallowedtoincubate.ColordevelopsinthepresenceofboundenzymeandtheopticaldensityisreadwithanELISAplatereader.

ELISA FormatsELISAsaredividedintothreemainformats—indirect,blocking(competitive)andantigen-capture(direct).

Indirect FormatIntheindirectformat,thesampleantibodyissandwichedbetweentheantigencoatedontheplateandanenzyme-labeled,anti-speciesglobulinconjugate.Theadditionofanenzymesubstrate-chromogenreagentcausescolortodevelop.Thiscolorisdirectlyproportionaltotheamountofboundsampleantibody.Themoreantibodypresentinthesample,thestrongerthecolordevelopmentinthetestwells.Thisformatissuitablefordeterminingtotalantibodylevelinsamples(Newcastlediseasevirus,B. abortus,etc.).

ELISA Technology

ELISA formats provide the ability to: • Test a large number of

samples at the same time

• Automate the procedure using robotics or other types of automated equipment

• Computerize the calculation and reporting of results

Indirect ELISA

Steps in an Indirect ELISA

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ELISABlocking (Competitive) FormatInthisformat,thespecificsampleantibodiescompetewith,orblock,theenzyme-labeled,specificantibodyintheconjugate.Theadditionofanenzymesubstrate-chromogenreagentcausescolortodevelop.Thiscolorisinverselyproportionaltotheamountofboundsampleantibody.Themoreantibodiespresentinthesample,thelesscolordevelopmentinthetestwells(CAV,CSFVAb,etc.).

Antigen-Capture (Direct) FormatIntheantigen-captureformat,theantigeninthesampleissandwichedbetweenantibodiescoatedontheplateandanenzyme-labeledconjugate.Theantibodyconjugatecanbeeithermonoclonalorpolyclonal.Theadditionofanenzymesubstrate-chromogenreagentcausescolortodevelop.Thiscolorisdirectlyproportionaltotheamountofthetargetantigenpresentinthesample(CSFVAg,LLAg,etc.).

Steps in Blocking ELISA

Blocking ELISA

ELISA Kits

AnELISAkitisasetofstandardizedreagentsandmicrowellplatesmanufacturedforaspecifictest.IDEXXELISAkitsmaycontainsomeorallofthefollowingcomponents:coatedplates(solidand/orstripplates),samplediluent,controls,washconcentrate,conjugate,substrateandstopsolution.Thekitsaremanufacturedinbatchesorlots.Eachcomponentofeachkitlotisoptimizedandmanufacturedtoworkasaunit.Thekitspassmanyquality-controltestsconductedbyIDEXX,numerousworldwidereferencelaboratories,and/ortheUSDAbeforetheyareapprovedandreleasedforsale.

NOTE:Donotmixorusecomponentsfromdifferentkitlotnumbers.

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ELISACoated Plates The96-wellplatesaremadeofpolystyreneandcoatedwitheitherinactivatedantigenorantibody.Thiscoatingisthebindingsitefortheantibodiesorantigensinthesample.Unboundantibodiesorantigensinthesamplearewashedawayafterincubation.

Forsomeassays,normalhostcell(NHC)antigensandpathogen-specificantigensarecoatedinalternatingrowsorcolumns.TheNHCantigensareusedtoassesswhetherantibodiesagainsttissueculturecomponentspresentinvaccinesarecontributingtosamplereactions.Whenusingtheseplates,eachsampleisaddedtobothasamplewellandanNHCwell.

Sample DiluentMostassaysrequireaspecificdilutionofthesample.Samplesareaddedtothesamplediluentandmixedpriortoputtingthemontothecoatedplates.

ControlsThepositivecontrolisasolutionthatcontainsantibodyorantigen.Thenegativecontrolisasolutionwithoutantibodyorantigen.Thecontrolshelptonormalizeorstandardizeeachplate.Controlsarealsousedtovalidatetheassayandtocalculatesampleresults.Inmostkits,thecontrolsarepredilutedandreadytouse.Besuretofollowtheinstructionsinthepackageinsert.

ConjugateELISAconjugatesareenzyme-labeledantibodiesorantigensthatreactspecificallytoplate-boundsampleanalytes.Unboundconjugateiswashedawayafterincubationandbeforetheadditionofsubstrate.Theopticaldensityofthecolorimetricsubstrateisdirectlyproportionaltothequantityofboundenzymepresent.

SubstrateForperoxidaseconjugates,thesubstrateisamixtureofhydrogenperoxideandachromogenthatreactswiththeenzymeportionoftheconjugatetoproducecolor.

Wash ConcentrateThewashconcentrateisabufferedsolutioncontainingdetergentusedtowashawayunboundmaterialsfromtheplates.

Stop SolutionThestopsolutionstopstheenzyme-substratereactionand,thereby,thecolordevelopment.

ELISA Kits continued

IDEXX kits are manufactured in batches or lots according to strict quality standards. Each component or reagent in a kit lot is optimized to work with the other reagents contained in the kit. This includes measurements of sensitivity, specificity and repeatability. Therefore, it is very important not to mix reagents from different kit lots.

All kit components have an expiration date.

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ELISAEquipmentforELISAtestingiswidelyavailable.Readers,washersandpipettesareavailableasmanualorautomatedsystems.Someofthefactorsaffectingequipmentselectionarethenumberandtypesoftestsandsamples,technicaltrainingofstaffandfinancialconsiderations.BelowisabriefoutlineofsomeequipmentavailableforperformingELISAtesting.

Pipettes• Single-channel,fixed-volumeandadjustable-volume(1–20µL,

10–100µL,20–200µL,etc.)

• Multichannel,8-and12-channels

• Semi-automateddispensingunits

• Fullyautomatedsystemsthatcanprocessmultipleplates

Dilutors• Single-channel

• Multichannel

• Automateddispensingunits

Washer Systems• Manualsystemsthatwashoneroworcolumnatatime

• Semi-automatedsystemsthathandleonestriporplateatatime

• Fullyautomatedsystemsthatcanprocessmultipleplates

ELISA Plate Readers• Manualreadersthatreadoneroworwellatatime

• Semi-automatedreadersthatreadoneplateatatime

• Fullyautomatedsystemsthatcanprocessmultipleplatessimultaneously

Other• Humiditychamber(notrequiredforallELISAtests)

• Platesealersforassaysthathavelongincubationtimes(toavoidevaporation)

ELISA Equipment

There is a large selection of equipment available. When purchasing a plate reader, call IDEXX Technical Services to make sure the xChek* software has the proper interface.

Multichannel pipette and single-channel pipette

Semi-automated wash system

Manual wash system

Plate reader

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ELISAThemaintenanceandcalibrationofyourlaboratoryequipmentisextremelyimportantinobtainingaccurateandreproducibleresults.

TheMaintenanceandCalibrationSchedule(AppendixD)shouldbeusedasaguideline.Adjustmaintenanceroutinesaccordingtotheamountofdailytestingperformedinyourlaboratory.Alwaysrefertoyourequipmentmanufacturer’sguidefortheirrecommendations.

Calibration ProtocolsEquipmentalwaysneedstobeinpropercalibration.Equipmentthatisoutofcalibrationcanproducefalseorinaccurateresults.RefertotheMaintenanceandCalibrationSchedule(AppendixD)andyourmanufacturer’sinstructionsforthepropercalibrationprotocolandrequiredfrequency.

Options for Calibrating Pipettes• PerformthegravimetricmethodoutlinedinAppendixA.

• UseacommercialautomatedcalibrationsystemlikethePCS®‚producedbyArtel.SeeAppendixA,GravimetricPipetteCalibrationProcedure,formoreinformation.

• Sendthepipettetothemanufacturer;seeyourowner’smanualforinstructions.

• Sendthepipettetoapipettecalibrationservice.

Sendingpipettesoutforserviceisbeneficialwhenrepairormaintenanceisnecessary.However,thispracticeprovidesonlyalimitedlevelofqualitycontrol,whichcanbeincreasedwithin-housecalibration.

Operatortechniqueandlaboratoryenvironmentaretwocriticalvariablesthatdeterminehowapipettewillperformwhenusedonyourbenchtop.Athoroughquality-controlprogrammustincludeaquantitativeaccountoftheseeffects.Itisbeneficialtohaveamethodinplacethatallowsyoutoperformregular,routineperformanceverificationsonyourownpipettes.Bydoingso,youwillbeabletotrackpipettesthataredriftingoutoftolerance.Whenthishappens,thefailingpipetteshouldbesentoutforcorrectivemaintenanceorrepairbyaqualifiedservicebeforeitcompromisesyourlaboratorydataandproductivity.

Equipment Maintenance and Calibration

Be sure to label your pipettes with the calibration date and keep a log for the calibration and maintenance of all your equipment.

Pipette with calibration label

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ELISAReceiving KitsWhenyoureceiveyourELISAkits,recordthedateonyourInventoryControlTrackingChart(AppendixB)andonthekitboxes.Inspectthemfordamageandstorethemat2°–7°C.Whenusingkitsfromyourinventory,usethefirst-in-first-out(FIFO)method.Inotherwords,usethekitsthataretheoldest(orwillexpire)first.Individualkitcomponentsmayhavelongerexpirationdatesthantheactualdateontheouterkitbox.However,youshouldgobytheexpirationdateontheouterlabelofthekit.Ifyoudonotuseanentirekit,markthedateitisopenedandeachtimeitisusedthereafter.Thisway,youcankeeptrackofhowmanytimesitmovesorcyclesfromtherefrigeratortoroomtemperature.Keepthenumberofcyclestoaminimumbybatching(oraccumulating)samplesintolargergroupswheneverpossible.

General Reagent HandlingBesuretocheckyourpackageinsertforguidelinesonhandlingandpreparingreagents.Sometestkitsrecommendthatallreagentsandplatesbebroughttoroomtemperature(18°–25°C)priortouse;othersindicatethatonlyspecificreagentsbebroughttoroomtemperature.Whenyouneedtobringakittoroomtemperature(18°–25°C),takeitoutoftherefrigeratorandtakethekitcomponentsoutofthekitboxatleast2–3hoursbeforebeginningtheassay.

Measureallreagentsusingsterileorcleanvessels.Becarefultomeasureonlywhatisneededforthenumberofplatesbeingrun.Thiswillhelptomaintaintheintegrityofthereagents.Donotreturnreagentstotheoriginalstockbottles.Westronglyrecommendusingdisposablepipettesandreservoirswhenhandlingreagentstominimizetheriskofcontamination.However,ifyouchoosetoreuseanydisposabledevice,useaseparatereservoirforeachreagentandbesuretolabelthem.Also,washandthoroughlyrinsethewellswithdeionizedordistilledwateraftereachuse.Changeanddiscardthedisposablereservoirsasfrequentlyaspossible.Neverusethesamereservoirforconjugateandsubstrate,evenifithasbeenwashed.

Reagent Handling and Preparation

The contamination of reagents may compromise your test results. Labeling your reagent reservoirs and using a separate one for each reagent will help minimize the risk.

Label your kit with the date it was received.

Labeled reservoirs

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ELISAPlatesMostassayplatesareprovidedinresealablebagsthatincludeadesiccant.Insomekits,theplatesarepackagedindividually.Whenopeningabagthatcontainsseveralplates,removeonlythenumberofplatesyouneed,resealthebagandreturnittotherefrigeratorforstorage.Ifpossible,theplatesthatweretakenfromtheoriginalbagshouldbestoredinanotherbagwithdesiccantsinit.Ifapartialplateisused,aspiratealltheliquidfromtheusedwellsandcoverthemwithsealingtape.Placeseveraldesiccantsinthebag.Besuretomarktheplateaccordinglyandstoreitseparatelyfromunusedassayplates.Somekitshavetheplatesformattedintostrips.Werecommendlabelingeachstripbeforeusetopreventconfusion.

Sample Diluent and Wash ConcentrateMakesurethesamplediluentandwashconcentratehavecometoroomtemperature(18°–25°C)beforeuse.Theseareusuallythelargestbottlesinakitandrequirethemosttimetoequilibrate.Ifthewashconcentratestillshowscrystalformationafterreachingroomtemperature,mixitbyinvertingitseveraltimes.

ControlsMostkitsareformulatedwithpredilutedcontrols.However,somerequirethatyoudilutetheminthesamemannerasyoursample.Controlsshouldbeaddedtotheplateinthesamemethodandatthesametimeasthesamples.

ConjugateIfthekitrequiresyoutopreparea“working”conjugatesolution,besuretofollowtheinstructionsclosely.Prepareonlywhatyouimmediatelyneed,anddonotsaveleftoversolutionforfutureuse.Ifconjugatesarecontaminatedorimproperlystored,theymayloseenzymaticactivityormayhaveanapparentincreaseinbackground.Mostkitssupplyaready-to-useconjugate.

SubstrateOurELISAtestkitsincludeaready-to-usesubstrate.Thechemicalactivityofthesubstratewillbecompromisedifitisexposedtolightorcomesintocontactwithmetal.Protectthissolutionbystoringitinadarkcontaineruntilreadyforuse.

Stop SolutionBesuretousethestopsolutionincludedinthekit.Followanysafetyprecautionsinthepackageinsert.Thestopsolutionshouldbeatroomtemperaturebeforeuse.Ifthestopsolutionshowscrystalformationafterreachingroomtemperature,mixitbyinvertingseveraltimes.Thestopsolutionmaycrystalizeatlowertemperatures.Beforeuse,makesurethatitiscompletelydissolvedandappearsclear.

Handling and Preparing Kit Components

Refer to the package insert for specific details on the kit you are using.

Do not exchange components between kit lot numbers, even if kits are of similar type. Test results may be severely and adversely affected.

Seal, label and store partially used plates in a bag with desiccant.

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ELISAIn-House ControlsWerecommendusingin-houseassaycontrolstomonitoryourELISAtechniquesandkitperformanceovertime.

Becauseseraaregenerallyreceivedinsmallquantities,controlswillneedtobemadebypoolingsamples.Collectnegativeandpositivesamplesseparately.Whensufficientquantitiesofeachhavebeencollected,poolsimilarsamplestogether.Mixthepooledsamplesthoroughly.Insmallquantities,performtheserialdilutionofpositiveserainnegativeserum.Assayeachdilutionaccordingtostandardkitprotocol(samesampledilutionasdescribedinthekitpackageinsert).Selectthedilutionthatismostcomparabletothesample-to-positive(S/P)orsample-to-negative(S/N)valuesthatyouwanttomonitor.Makelargequantitiesofthatdilution.Prefilterthepreparedcontrolsusinga0.45-micronfiltermembrane;youmaythenchoosetofilterwitha0.20-micronfiltermembrane(optional).Putasmallamountofthefreshlymadein-housecontrol(volumeenoughfor1–2tests)intoairtightvials,label,dateandstorefrozenat-70°Cifpossible.Keeparecordofthispreparationinanotebookforreference.

Tousethiscontrol,thaw,mixanddiluteitinthesamemannerasaroutinesample.Runitoneveryplatenexttothekitcontrols.Donotrefreezeyourin-housecontrol.Youcanstoreitfor3–5daysat4°C.

RecordyourresultsontheLaboratoryTrackingChart(AppendixC)andgraphthem.Anyvariationsortrendsshouldalertyoutoreviewyourtechniqueandquality-controlmeasures.

Monitoring TemperatureELISAtestsaresensitivetotemperatureextremes.Trytomaintainalaboratorytemperatureof18°–25°C.Avoidrunningassaysunderornearairventsasthismaycauseexcessivecooling,heatingand/orevaporation.Also,donotrunassaysindirectsunlightasthismaycauseexcessiveheatandevaporation.Coldbenchtopsmayaffectyourassayandshouldbeavoidedbyplacingseverallayersofpapertowelsorsomeotherinsulatingmaterialundertheassayplatesduringincubation.

Recordandtrackthetemperatureduringeachassay.Ifyourlaboratory’stemperaturefluctuatesfrommorningtoafternoon,recordthisonyourtrackingchart.Ifyouhaveconditionsthataredifficulttocontrol,itisagoodideatouseatemperaturecontrolchambertoincubateyourplates.UsingELISAplatecoverswillhelpcontrolevaporationandaccidentalspills.

Quality Control CheckUsetheQualityControlQuickCheck(AppendixE)totroubleshootanyproblems.

Quality Control

To help troubleshoot questionable results, record and graph the laboratory temperature.

IBV Control Tracking September

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Tracking chart for controls

Laboratory temperature tracking chart

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ELISAIncoming Sample QualitySamplequalitycanhaveasignificantimpactonfinalassayresults.Mostlabshavenochoiceregardingthequalityofincomingsamples.Inmanycases,thesamplediluentformulationcompensatesforvariationsinsamplequality.

Grossfungalorbacterialcontaminationcanhaveadverseeffectsontheantibodyorotherproteincomponentsofasampleandmayhaveanundesirableeffectontestresults.Ifsamplequalityishighlyquestionable,obtainingafreshsampleisstronglyadvised,whenpossible.

Serum/Plasma SamplesSerumsampleswithtracehemolysis(light-redcolor)andmoderatelipemia(milkyappearance)mayhavelittleornoeffectonELISAresults.Avoidusingsamplesthatareheavilyhemolyzed(dark-redcolor)orgrosslylipemic.Checkyourpackageinsertforinformation.Whenserumisontheclot,becarefulnottoaspirateanyoftheclottedmaterialorbloodcells.

Meat Juice SamplesMeatjuicesamplesshouldbeascleanaspossible.Removedebrisandlipidsfromthesamplewhenpipetting.

Milk SamplesWholemilksamplescanbeusedaftercentrifugationfor15minutesat2000xg,orleftovernightifrefrigerated(2°–8°C).Thesampleintendedfortheassayshouldbedrawnfrombelowthecreamlayer.

Egg Yolk SamplesCollectsampleswithacleantuberculinsyringeandmixthedilutedsamplesthoroughlybyvortexing.

Other Sample TypesRefertoyourpackageinsertforsamplehandling,preparationandstorageofothersampletypes(e.g.,albumin,cloacalswabs).

Meat juice sample Take sample from the area indicated.

Blood sample Take sample from the area indicated.

Milk sample Take sample from the area indicated.

Sample Handling

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ELISA

Light hemolysis

Dark hemolysis

Unmixed thawed sample Proteins settled on bottom of tube; mix prior to taking sample

Avoid numerous freeze-thaw cycles, as this may damage the antibodies or antigens in the sample. We recommend no more than 3–5 cycles.

Storing SamplesBesuresamplesareproperlystored.Ingeneral,serumsamplesshouldberefrigeratedat2°–7°Cfornomorethan3–5days.Ifsamplesneedtobestoredforalongerperiod,theyshouldberemovedfromtheclotandfrozentoaminimumof-20°C.Makesureallstoredsamplesareproperlylabeledandsealedtopreventevaporation.Evidenceoflyophilization(concentrationofthesample)canbeseenascrystallizationandiscommoninself-defrostingfreezers.Thisshouldbeavoidedbecausetheintegrityofthesamplewillmostlikelybecompromised.

Using Frozen SamplesFrozensamplescanbethawedatroomtemperatureorinarefrigerator.Allthawedsamplesneedtobethoroughlymixedpriortodilutiontoensurethattheproteinsaredispersedthroughoutthesample.Mixbygentlevortexingorinvertingatleastfivetimes.Frothingorover-mixingofsampleswillcausedenaturationofserumproteins.

Sample Handling continued

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ELISATwopipettingmethodsusedforELISAarestandard(forward)andreverse.Notallpipettesarecapableofreversepipetting.Refertotheinstructionsincludedwithyourpipettefordetails.

Usestandard(forward)pipettingforthepreparationofsampledilutions,andreversepipettingfortheadditionofdilutedsamples,controlsandreagents.

Carefulpipettingiscrucialforaccuratetestresults.Becomefamiliarwiththepipetteandbothmethodsbeforerunningactualtests.Besuretousethecorrectpipetteandtip(volumecapacity)forthevolumebeingtransferred.

Pipetting Technique1. Drawthecalibratedvolumeofsampleintothetip.

2. Touchthesideofthetubewiththetiptoremovetheexcessliquid.

3. Ensurethatyouhavethepropervolumeofsampleinthetip.

Whenusingamultichannelpipette,ifthewellsonyourplateareempty,positionthetipsintothelowercornerofeachwell,makingcontactwiththeplastic.Ifthewellsonyourplatecontainliquid,positionthetipsabovetheliquid,makingcontactwiththeplastic.

Use standard (forward) pipetting for the preparation of sample dilutions, and reverse pipetting for the addition of diluted samples, controls and reagents.

Pipetting a Sample

Proper Pipetting

Draw up the calibrated volume of sample into the tip.

The drop on the tip needs to be removed.

Touch the side of the tube with the tip to remove the excess liquid.

Ensure that you have the proper volume of sample in the tip.

Proper position to dispense reagents into empty wells using a multichannel pipette; in the lower corner of each well

Proper position to dispense reagents into wells containing liquid using a multichannel pipette; above the liquid

Pipetting Methods

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ELISAStandard (Forward) Pipetting and Sample Preparation1. Putanewtiponasingle-channelpipetteandmakesurethatitisontight.

2. Presstheplungertothefirststop.

3. Somemanufacturersrecommendthatyouprewetthetipbyaspiratingandexpellinganamountofthesample.Checktheinstructionsthatcamewithyourpipette.

4. Drawthecalibratedvolumeofsampleintothetipandpauseforonesecondwiththetipstillinthesample.Becarefulnottoplacethetiptoodeeplyintothesample.

5. Touchthetiptothesideofthesamplecontainertoremoveanyexcessliquidontheoutsideofthetip.

6. Dispensethesampleintothemeasureddiluentbydepressingtheplungerpastthefirststoptothesecondstop.Becarefulnottoplacethetiptoodeeplyintothesamplediluent.

Forsampleslessthanorequalto10µL:Afterdispensingthesampleintothediluent,rinsethepipettetipinthediluentbypushingtheplungerdown2–3timesbeforeejectingthetip.

7. Mixsampleswithamultichannelpipettepriortodispensingsamplesontotheplate.Youcandothisbypushingtheplungerdown3–6times.

8. Ejectthetipintoawastecontainer.

Reverse Pipetting Using a Multichannel Pipette1. Putnewtipsonthepipette.Makesuretheyareontightandstraight.

2. Presstheplungerpastthefirststopandhalfwaytothesecondstop.

3. Drawtheliquidinaslowmotion,beingcarefulnottodrawanyairbubblesintothetips.Checkforconsistencyofvolumeinthetips.

4. Touchthetipstotheedgeofthereagentreservoirtoremoveexcessliquidontheoutsideofthetips.

a. Ifthewellsonyourplateareempty,positionthetipsintothelowercornerofeachwell,makingcontactwiththeplastic.

b. Ifthewellsonyourplatecontainliquid,positionthetipsabovetheliquid,makingcontactwiththeplastic.

5. Slowlydispensetheliquidintothewellsbydepressingtheplungertothefirststop.Becarefulnottosplashliquidoutofthewells,andmakesuretherearenodropsleftonthetips.

6. Torepeat,holdtheplungeratthefirststopandcontinuewithstep3.

7. Ejectthetipsintoawastecontainer.

NOTE:Reversepipettingusesmorereagent/volume(=“deadvolume”).

Bent tips; needs to be replaced

Pipetting Methods continued

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ELISAAutomated Dilution Systems and Competitive AssaysForthosesystemsandassaysusingneatsamplesorlowerdilutionfactors,thesamplecanbeputdirectlyintothewellsofthecoatedplates.

Followthesequencebelow:

1. Addthediluenttotheplate.

2. Addthesampleintothediluent.

3. Mixbytappingtheplateorrepeatingpipetting.

Automated equipment uses more reagent/volume than semiautomated. Check your manufacturer’s recommendations for purging and priming your system.

Pipetting Methods continued

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ELISAAdding Samples and ControlsIncubationsforassayplatesshouldbetimedaspreciselyaspossible.Usuallytheprocessofaddingsamplestotheplaterequiresthemosttime.Whenyoudispensesamplesontotheplate,itiscriticaltokeepthetimedifferencebetweenthefirstandthelastsampletoaminimum.

Useamultichannelpipettewheneverpossibletominimizethetimeintervalfromthestartoftheplatetotheend.Ifthetimeintervalbecomestoolongoryouareinterruptedwhileaddingsamplestotheplate,putapositivecontroland/oryourin-housecontrolattheendoftheplateandcomparetheseresultswiththecontrolsatthebeginningoftheplate.

Fortightercontroloverthetimedifferentiationfromwhenthecontrolsandsamplesareadded,youcanputyourcontrolsinatubethatisrackedinpositionwithyoursamples.Thenuseamultichannelpipetteandputthecontrolsontotheplateatthesametimeyouareaddingthesamples.

Multiple Plate RunsWhentimingmultipleplates,itisimportanttokeeptrackofthetimeintervalfromthefirstplatetothelastplateintherun.Keepyourbatchsizessmallenoughsoyourprocessesdonotoverlap.Youdonotwanttobewashingaplate(unlessitisdonebyanautomatedwasher)whileanotherneedstohaveconjugateadded.Ifpossible,useatimerforeveryplate.

To minimize incubation time between controls and samples, rack the controls with the samples and add them to your plate using a multichannel pipette.

Use several timers when incubating multiple plates.

ELISA Plate Timing

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ELISAAutomated or Semiautomated SystemsIngeneral,anautomatedorsemiautomatedwashsysteminproperworkingorderwillprovidemoreconsistentwashingthanmanualmethods.Checkthatallthedispensingneedlesaredispensingwithasmooth,steadystreamandthatallaspirationportsaspirateuniformly.

Makesureyourwashsystemisproperlycleanedandmaintained.RefertotheEquipmentMaintenanceandCalibrationsectioninthisguide(page6)andyourowner’smanualforpropermaintenance.Theplate-washingtechniqueshouldbeconsistentfromplatetoplateandfromrowtorowwithinaplate.Avoidprolongedsoaktimesunlessspecificallyrecommendedinthepackageinsert.

Preparethewashsolutionaccordingtothepackageinsert.Useonlythewashsolutionincludedwith,orrecommendedfor,yourkit.

Aspiratereagentsfromtheplatebeforedispensingthewashsolution.

Followthespecificrecommendationsinyourpackageinsertforthenumberofwashestouseateachstepofanassay.Mostassaysrequireapproximately300–350µLperwellperwash.Becarefultofillthewellsabovethelevelofthereagents.Donotallowwellstooverflow.Ifthisoccurs,thetestresultsmaybeinvalid.

Donotallowtheplatetodrybetweenplatewashingsandpriortotheadditionofreagents.

Afterthefinalaspiration,tapoutanyremainingliquidontoseverallayersofpapertowels.

Whentestingmilk,albumin,wholebloodoryolksamples,takeextracaretoinspectthewells.Becauseoftheirproteinorfatcomposition,thesesampletypesaresometimesmoredifficulttowashfromthewellsandmayrequirethemaximumrecommendednumberofwashcycles.

After tapping out plates, check paper towels for any evidence of color. This may indicate that the plates were not washed properly and there are reagents remaining in or around the wells.

Microbial growth in wash system tube; needs to be replaced

ELISA Plate Washing

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ELISAManual or Semi-Manual SystemsWorkquicklysothetimefromwashingthefirstwell/rowtothelastisminimal.Ifthetimeistoolong,theemptywellsmaydryoutandthelastwellswillhavealongerincubationthanthefirstwells.

Washingbymultichannelpipetteorwashbottlesshouldbeavoidedifatallpossibleasthisgivesinsufficientwashingthatresultsinhighandinconsistentbackground.

Makesuretoaspiratealltheliquidfromthewellsbyplacingtheaspirationneedlesatthebottomandinthecornersofthewells.Donotscrapethesurfaceoftheplateasthiswillremovetheantigen/antibodyboundtothesurfaceandcauseinconsistentorinaccurateresults.Afteraspiration,wellsshouldnotdrybeforetheadditionofthenextreagent.

Aftertappingouttheplates,checkthepapertowelsforanyevidenceofcolor.Thismayindicatethattheplateswerenotwashedproperlyandtherearereagentsremaininginoraroundthewells.

Overflowing plate; this can contaminate other wells

Proper position of manual washer needles for dispensing wash solution

Proper position of manual washer needles for aspirating liquid

ELISA Plate Washing continued

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ELISAReading PlatesThelaststepinanELISAistoreadandinterprettheresults.Formostassays,theopticaldensity(amountofcolor)ofthesolutionontheplateisreadwithaspectrophotometer,commonlyknownasaplatereader.Therearemanymodelsandmanufacturersofplatereaders;refertothemanufacturer’sinstructionsfordetailsofoperation.

Thepackageinsertspecifieswhichwavelengthisrequiredfortheassay.Mostassaysspecifytheabsorbancereadingat450nmor650nm.Mostassaysareoptimizedusingaplatereaderequippedwitha650-nmfilter.Otherfilterscanbeused,butwillresultinloweropticaldensity(OD)values.Theuseof630-nmor620-nmfilterswilllowertheODvaluesofboththecontrolsandsamples,butwilldosoequivalentlyacrosstheentireplate.Theuseofthesealternativefilterswillnotaffectthetestresults.

Platesshouldbereadassoonaspossiblefollowingtheadditionofstopsolution.Absorbancereadingsmaydriftifexcessivetimeelapsesbetweenstoppingthereactionandreadingtheplates.

Data ManagementIDEXXprovidesthexChek*softwaretoassistyouinthecollectionandmanagementofthedatafromyourELISAassays.ThexCheksoftwareinterfaceswithmostcommonplatereaderstoreadtheplate,sendtheopticaldensitiestothecomputerandcalculatetheresults.AnIDEXXTechnicalServicesRepresentativecanassistyouinlearningmoreaboutthissoftware.

The xChek* software interfacing with the ELISA plate reader

Reading Plates and Data Management

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ELISAThisinformationisintendedtohelpyoutroubleshootyourELISAprocedure.Ifyouneedassistance,contactIDEXXProductionAnimalServicesTechnicalServicesat1-207-556-0895,option2;or1-800-548-9997,option2.

NOTE:TheconditionsdescribedheremaynotpertaintoeveryELISAkitbecauseperformancerequirementsvaryforindividualassays.Besuretocheckyourpackageinsertforspecifications.

ELISA Troubleshooting

Issue:High background or excessive color development (high optical density [OD] readings)

Possible Causes Recommended Actions

Poor-qualitywaterwasusedtowashplatesortoprepare

washsolution.

Checkthewaterquality.Ifitisquestionable,trysubstitutinganalternatewatersource,suchasbottleddistilledwater,towashplatesorpreparethewashsolution.

Substratesolutionhasdeteriorated.

Makesurethesubstrateiscolorlesspriortoadditiontotheplate.

Therewasinsufficientwashingorpoor

washerperformance.

Tryusingthehighestnumberofwashesrecommendedfortheassay.Makesurethatatleast350µLofwashsolutionisdispensedperwellperwash.Verifytheperformanceofthewashersystem.Havethesystemrepairedifanyportsdrip,dispenseoraspiratepoorly.

Washersystemhadmicrobialcontamination.

Cleanoutmicrobialcontaminationbyflushingthesystemwithadilutesolutionofbleach(10%byvolume)followedbyalargeamountofdistilledordeionizedwater.Primethesystemwiththeappropriatewashsolutionbeforeuse.Thetubingmayneedtobechangedifthecontaminationisheavy.

Washsystemcontainedanalternatewashformulation.

Besureeachuniquewashsolutionisproperlylabeled.Primethesystemthoroughlywhenswitchingbetweensolutions.

Readerwasmalfunctioningornotblankedproperly;

thisisapossiblecauseiftheODreadingswerehighand

thecolorwasnotdark.

Verifythereader’sperformanceusingacalibrationplateandcheckthelampalignment.Verifytheblankingprocedure,ifapplicable,andreblank.

Laboratorytemperaturewastoohighortoolow.

Maintaintheroomtemperaturewithin18°–25°C.Avoidrunningassaysnearheatsources,indirectsunlightorunderairvents.

Reagentswereintermixed,contaminatedor

preparedincorrectly.

Ensurethatthecorrectreagentswereused,thatworkingsolutionswerepreparedcorrectlyandthatcontaminationhasnotoccurred.

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ELISAIssue:Insufficient color development (low optical density [OD] readings)

Possible Causes Recommended Actions

Laboratorytemperaturewastoolow.

Maintaintheroomtemperaturewithin18°–25°C.Avoidrunningassaysunderairconditioningventsornearcoldwindows.

Washsolutionwaspreparedincorrectlyorthewrongwash

solutionwasused.

Besuretousethewashsolutionrecommendedforthekitandthatitispreparedcorrectly.Labeleachuniquewashsolutiontoavoidusingthewrongone.

Washersystemhadmicrobialcontamination

orcontainedanalternatewashformulation.

Cleanoutmicrobialcontaminationbyflushingthesystemwithadilutesolutionofbleach(10%byvolume)followedbyalargeamountofdistilledordeionizedwater,thenprimethesystemwiththeappropriatewashsolution.Besureeachuniquewashsolutionisproperlylabeled.Primethesystemthoroughlywhenswitchingbetweensolutions.

Toomanywashcycleswereused.

Staywithintherecommendedrangeforthenumberofwashcycles.Trytousethelowestnumberofwashesrecommendedfortheassay.

Incubationperiodsweretooshort.

Followprotocolforincubationtimes.Timeeachplateseparatelytoensureaccurateincubationperiods.

Reagentsandplatesweretoocold.

Makesureplatesandreagentsareatroomtemperaturebytakingthemoutoftherefrigerator,andthekitcomponentsoutofthekitbox,atleast2–3hoursbeforestartingtheassay.

Reagentswereexpiredorintermixedfromadifferent

lotnumber.

Verifytheexpirationdatesandlotnumbersonthereagents.

Wrongconjugatewasused,conjugatewas

preparedincorrectlyorhasdeteriorated.

Besurethattheconjugateusedistheonethatcamewiththekitandallconjugatesarekit-andlot-specific.Ifpreparationofaworkingconjugateisneeded,besurethattheconcentrateanddiluentaremixedinappropriatevolumes.Donotpreparetheworkingsolutiontoofarinadvance,anddonotsaveanyunusedportionforfutureuse.Ifnoconjugatepreparationisnecessary,besuretopouroutonlytheamountrequiredforimmediateuse,anddonotreturnanyunusedportiontothestockbottle.

Assayplatereadwasatwrongwavelength,orreader

wasmalfunctioning.

Verifythecorrectwavelengthfortheassayandreadtheplateagain.Verifyreadercalibrationandlampalignment.

Positivecontrolwasdiluted(indirectformatonly).

Donotdilutecontrolsunlessspecifiedinthepackageinsert.

Excessivekitstresshasoccurred.

Checkrecordstoseehowmanytimesthekithascycledfromtherefrigerator.Checktoseeifthekitwasleftoutonaloadingdockorotherareafortoolongoratextremetemperatures.

Assayplateswerecompromisedorpreviouslyused.

Besuretorefrigerateplatesinsealedbagswithadesiccanttomaintainstability.Preventcondensationfromformingonplatesbyallowingthemtoequilibratetoroomtemperaturewhileinthepackaging.Ifpartialplatesareused,besuretolabelusedwellstopreventreuse;coverthemwithsealingtapeandusetheremainingwellsassoonaspossible.Donotstorepartiallyusedplateswithotherplates.Includeadesiccantinthestoragebag.

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ELISAELISA Troubleshooting continued

Issue:Replicates within a plate show poor reproducibility

Possible Causes Recommended Actions

Excessivetimewastakentoaddsamples,controlsorreagentstotheassayplate.

Besuretohaveallmaterialssetupandreadytousequickly.Useamultichannelpipettetoaddreagentstomultiplewellssimultaneously.Rackcontrolswithsamplesanddispensethemontotheplateatthesametimeasthesamples.

Multichannelpipettewasnotfunctioningproperly.

Verifypipettecalibrationandcheckthattipsareontight.Besureallchannelsofthepipettedrawanddispenseequalvolumes.

Therewasinconsistentwashingorwasher

systemmalfunctioning.

Verifytheperformanceofthewashersystem.Havethesystemrepairedifanyportsdripordispense/aspiratepoorly.

Therewaspoordistributionofantibodyinthesample.

Ifthesamplewasthawedorrefrigerated,makesureitwasmixedpriortodilution.Dilutedsamplesalsoneedtobemixedpriortoaddingthemtotheplate.

Issue:No color development

Possible Causes Recommended Actions

Reagentswereusedinthewrongorderoranassaystep

wasomitted.

Checkthepackageinsertfortheassayprotocolandrepeattheassay.

Sampleswerenotaddedtodiluent(indirectformatonly).

Verifythatthesampleswereaddedtothediluent.

Wrongconjugatewasused,conjugatewas

preparedincorrectlyorhasdeteriorated.

Besurethattheconjugateusedistheonethatcamewiththekit.Allconjugatesarekit-andlot-specific.Ifpreparationofaworkingconjugateisneeded,besurethattheconcentrateanddiluentaremixedincorrectvolumes.Donotpreparetheworkingsolutiontoofarinadvanceanddonotsaveanyunusedportionforfutureuse.Ifnoconjugatepreparationisnecessary,besuretopouroutonlytheamountrequiredforimmediateuseanddonotreturnanyunusedportiontothestockbottle.

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ELISAELISA Troubleshooting continued

Issue:Poor reproducibility plate to plate

Possible Causes Recommended Actions

Inconsistentincubationtimesoccurredfromplatetoplate.

Timeeachplateseparatelytoensurethatplateshaveconsistentincubationperiods.

Inconsistentwashingoccurredfromplatetoplate.

Usethesamenumberofwashesforeachplate.Verifytheperformanceofthewashersystem.Havethesystemrepairedifanyportsdripordispenseoraspiratepoorly.

Pipettewasworkingimproperly.

Checkthepipettecalibration.Verifythatpipettetipsareontightbeforeuseandthatallchannelsdrawanddispenseequalvolumes.

Kitcontrolsandsampleswereatdifferent

temperatures.

Besuretoallowsufficienttimeforsamplediluent,samplesandkitcontrolstocometoroomtemperaturebyremovingthemfromthekitbox.Largervolumeswillrequirelongerequilibrationtime.Ifusingawaterbathtohastenequilibration,makesureitismaintainedatroomtemperature;donotuseawarmwaterbathforcontrols,samplesordiluent.

Reagentswerebeingusedfromdifferentkitlots.

Ifrunningtwodifferentkitlotsatthesametime,makesuretolabelreagenttrays,etc.,soallreagentswithinalotareusedwiththecorrespondingplates.

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ELISAFor adjustable-volume pipettes, calibrations should be performed at a minimum of two settings—a low volume and a high volume at commonly used settings.

Materials

•Pipette •AnalyticalBalance •GlassBeaker

•DeionizedorDistilledWater •WeighingVessel •Thermometer

Procedure1. Toavoiderroneousresultsduetoevaporation,werecommend

humidifyingtheanalyticalbalancechamberatleast2hourspriortocalibration.Thiscanbeachievedbyplacingasmall,half-filledbeakerofwaterintothechamberwithallitsdoorsclosed.

2. Followthemanufacturer’sdirectionsforcleaningandlubricationofpipettespriortocalibration.

3. Theroomtemperatureshouldremainconstantwithin±0.5°C,preferablybetween18°–25°C.

4. Allowasufficientvolumeofdeionizedordistilledwatertocometoroomtemperatureandtakeatemperaturereading.

5. Recordthebeginningweightoftheweighingvesselorzerothebalance.

6. Usinganewpipettetipwitheachdelivery,pipettewaterintotheweighingvesselandrecordtheweight.Repeatthisstep10times.

7. Calculatethevolumedispensedforeachdelivery.

Calculations1. Calculatetheactualvolumedeliveredasfollows:

Volume= WeightofWater

DensityofWater

Densityofwaterat16°–21°C=0.998mg/µL Densityofwaterat22°–25°C=0.997mg/µL

2. Calculatethemean(M),standarddeviation(SD)andcoefficientofvariance(CV)ofthe10volumestodetermineprecisionofthepipette.

3. Determinetheaccuracyofthepipetteasfollows:(1-[Differencebetweenstatedandactualvolume/statedvolume])X100=%accuracy

Recommended specifications1. Precision:CV≤5.0%

2. Accuracy:≥95%

LabelingLabelthepipettewiththecalibrationdate,thetechnician’sinitials,theprecisionandtheaccuracy.Also,recordthesedatainalaboratorynotebookorlogforlong-termstorage.

Appendix A: Gravimetric Pipette Calibration Procedure

Automated Calibration SystemThe ARTEL PCS® Pipette Calibration System is an automated instrument/reagent system designed to facilitate regular and frequent verification of pipette performance and operator technique. For more information call ARTEL, at 1-888-406-3463 or 1-207-854-0860, or visit their Web site at artel-usa.com.

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Date

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Kit Lot Number

Plate Numbers

Negative

Control

Positive Control

Internal Control

Internal Control

Internal Control

Internal Control

Room Temperature

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ELISAAppendix D: Maintenance and Calibration Schedule

Procedure Daily Weekly Monthly Quarterly Yearly

Pipettes

Cleanexterior X

Checkcalibration X

CleaninteriorandO-rings X

Dilutor

FlushsystemwithDIwater X

Purgesystem X

Checkcalibration* X

Soaksyringes X

Check/Changetubings Check Change

Washer

FlushsystemwithDIwaterwhenusingwashsolutionsotherthanDIwaterandwhenchangingbetweenwashsolutiontypes

Checktraps,filtersandfoaming X

Checkaspirationanddispensingneedlesfordripsanddebris

Checktubesandbottlesformicrobialgrowth X

Decontamination—flushsystemwithbleachoralcoholsolution* X

Calibrationcheck—Purgesystem X

Cleanexterior X

Runthrough“cleancycle”withDIwater*

Check/Changetubing Check Change

Reader

Calibrationplate** X

Lampalignment Afterbulbreplacement

Cleanoptics X

Cleanexterior X

*Refertoyourmanufacturer’sguideforspecificinstructionsforyourmakeandmodel.**CallIDEXXTechnicalServicesoryourreadermanufacturerforrecommendationsoncalibrationplates.

X

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ELISAMonitoringandtrackingassayperformanceonquality-controlchartsprovidesinsightastowhenitisnecessarytotroubleshootproblems.BelowisachecklisttoreviewifyouarehavingproblemswithyourELISA.Ifyouhavefollowedallthestepsbelowandarestillhavingproblemswithyourassay,contactIDEXXProductionAnimalServicesTechnicalServicesat1-207-856-0895,option2;or1-800-548-9997,option2.

✔ EquipmentmKeeppreventivemaintenanceup-to-date

mCalibrateandcleanpipettes

mCalibratereader

mSanitizeandmaintainwashsystem

✔ ReagentsmMaintaininventorycontrol—FIFO

mInspectcomponents

mBringreagentstoroomtemperature

mAvoidcontamination

mVerifyproperstorage

✔ TechniquemMonitorsamplequality

mVerifyreagentpreparation

mVerifyappropriatesamplemixing

mVerifyproperpipetting

mChecktiming—multipleplateruns

mCheckwashingofassayplates

mUsein-housecontrols—trackresults

✔ OthermMonitorlaboratorytemperature

mUsesteriledisposablesandreservoirs

mMonitorassayperformance;recordinalog

Appendix E: Quality Control Quick Check

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ELISANotes

ELISA Technical Guide

i d e x x . c o m