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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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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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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.
ELISA Troubleshooting continued
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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.
Ap
pe
nd
ix B
:
Inve
nto
ry C
on
trol
Tra
ck
ing
Ch
art
Kit
Typ
eLo
t N
umb
erN
umb
er o
f Kits
Rec
eive
dD
ate
Rec
eive
dE
xpir
atio
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ate
C
om
men
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ELISAA
pp
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dix C
: Flo
ck
Ch
ek
*, He
rdC
he
k* a
nd
CH
EK
IT* L
ab
ora
tory T
rack
ing
Ch
art A
ssayNam
e:
Co
mm
ents
*FlockChek,H
erdChekandC
HE
KITaretradem
arksorregisteredtrademarksofID
EXXLaboratories,Inc.intheU
nitedStatesand/orothercountries.
Date
Technician
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
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