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AntibodiesOnline/Human Th1/Th2 Array Q1/ABIN625756/50 samples
Reactivity
Human
MethodType
SandwichELISA
Application
AntibodyArray(AA),MultiplexELISA(mpELISA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeQuantibody®HumanTh1/Th4Array1Kit.Detects10HumanTh1andTh4Cytokines.Suitableforallliquidsampletypes.
BrandQuantibody®
SampleTypeBodyFluids,CellCultureSupernatant,Plasma,Serum,CellLysate,TissueLysate
AnalyticalMethodQuantitative
DetectionMethodFluorometric
SpecificityGM-CSF,IFN-gamma,IL-10,IL-13,IL-2,IL-4,IL-5,IL-6,IL-8(CXCL8),TNFalpha
Characteristics
  • RunninganarrayislikerunningdozensofELISAssimultaneously.
  • Quantibodyarraysarestunninglysimpletouse,read,andanalyze.
  • Eachpanelcanquantifyupto40differentbioMarkerssimultaneously,andindividualpanelscanbemultiplexedtoquantifyasmanyas660differentbiomarkersatonetime.
  • Theentireprocesscanbecompletedinjust4-6hours.
  • Morecost-effectivethantraditionalELISA
  • Highspecificityandsystemreproducibility
  • Suitablefordiversesampletypes
  • Lowsamplevolumerequirement:50μLorless
  • Well-suitedforhighthroughputassays

    • Morecost-effectivethantraditionalELISA
    • Highspecificityandsystemreproducibility
    • Suitablefordiversesampletypes
    • Lowsamplevolumerequirement:50μLorless
    • Getresultssameday(6-hourprocessingtime)
    • Well-suitedforhighthroughputassays
    • QAnalyzersoftwareprovidesone-stepcomputation
ComponentsGlassChipwithantibodyarrays
SampleDiluent
Lyophilizedproteinstandardmix
Detectionantibodycocktail
Streptavidin-Fluorescentdye
Washbuffer
MaterialnotincludedDistilledordeionizedwater
Smallplasticboxesorcontainers
Pipettors,pipettetipsandothercommonlabconsumables
Orbitalshakeroroscillatingrocker
Aluminumfoil
Genemicroarrayscannerorsimilarlaserfluorescencescanner
ApplicationNotesCompletelycoverarrayareawithsampleorbufferduringincubation.Avoidfoamingduringincubationsteps.Performallincubationandwashstepsundergentlerockingorrotation.Covertheincubationchamberwithadhesivefilmduringincubation,particularlywhenincubationismorethan2hoursor<70 μLofsampleorreagentisused.Severalincubationstepssuchasstep6(blocking),step7(sampleincubation),step10(detectionantibodyincubation),orstep13(Cy3equivalentdyestreptavidinincubation)maybedoneovernightat4 °C.Pleasemakesuretocovertheincubationchambertightlytopreventevaporation.
Comment

TheQuantibodyarraysarequantitativemultiplexELISAarraysfeaturingfluorescentdetection.Theantibodiesarespottedonglassslidesolidsupportsandrequirealaserscannerfordatacollection.Cytokinestandardsareprovidedwiththearrayforcalculationoftargetproteinconcentrations.
AllQuantibodyarraysfeaturethesandwichimmunoassayprinciple,utilizinganimmobilizedcaptureantibodyalongwithacorrespondingbiotinylateddetectionantibody.

SampleVolume100μL
AssayTime6h
PlateGlassSlide
Protocol
  1. EachQuantibodyarraystartswithasingleglassmicroscopeslide,whichactsasasupportforthearray.Slidesaresegmentedusingarubbergasket.Upto8samplesmayassayedusingasingleslide.
  2. Antibodiesagainstavarietyofdifferentantigens(upto40biomarkersperslide)areprintedontotheglassslide.Replicatesareincluded,savingyoubothtimeandprecioussamplevolume.
  3. Theend-useraddseitherknownconcentrationstandards(included)oraqueoussampletoeachwellontheslide.Antibodiesontheslidecaptureantigenofffromthesampleorstandard.
  4. Theend-useraddsadetectionmixcontainingpairedantibodies(compatiblewiththeprimariespre-coatedontheslide)conjugatedtoafluorescentdyefordetection.
  5. Fluorescentsignalfromeachspotisreadusingalaserslidescanner.Theintensityfromeachspotiscomparedtothestandardcurve,andaquantitativeexpressionprofileforrelevantbiomarkersisestablished.
SamplePreparation

Useserum-freeconditionedmediaifpossible.Ifserum-containingconditionedmediaisrequired,itishighlyrecommendedthatcompletemediumbeusedasacontrolsincemanytypesofseracontainscytokines.Werecommendthefollowingparametersforyoursamples:50to100loforiginalordilutedserum,plasma,cellculturemedia,orotherbodyfluid,or50-500g/mlofproteinforcellandtissuelysates.Ifyouexperiencehighbackgroundorthereadingsexceedthedetectionrange,furtherdilutionofyoursampleisrecommended.

AssayProcedure
  1. Takeouttheglassslidefromthebox,andletitequilibratetoroomtemperatureinsidethesealedplasticbagfor20-30minutes.Removeslidefromtheplasticbag,peeloffthecoverfilm,andletitairdryforanother1-2hours.
    2.ReconstitutetheCytokineStandardMix(lyophilized)byadding500µlSampleDiluenttothetube.Forbestrecovery,alwaysquick-spinvialpriortoopening.Dissolvethepowderthoroughlybyagentlemix.LabeledthetubeasStd1.
    3.Label6cleanmicrocentrifugetubesasStd2toStd7.Add200µlSampleDiluenttoeachofthetubes.
    4.Pipette100µlStd1intotubeStd2andmixgently.Perform5moreserialdilutionsbyadding100µlStd2totubeStd3andsoon.
    5.Add100µlSampleDiluenttoanothertubelabeledasCNTRL.DonotaddstandardcytokinesorsamplestotheCNTRLtube,whichwillbeusedasnegativecontrol.Forbestresults,includeasetofstandardsineachslide.
    6.Add100µlSampleDiluentintoeachwellandincubateatroomtemperaturefor30minutestoblockslides.
    7.Decantbufferfromeachwell.Add100µlstandardcytokinesorsamplestoeachwell.Incubatearraysatroomtemperaturefor1-2hour.
    8.Wash:
    -Decantthesamplesfromeachwell,andwash5times(5mineach)with150µlof1XWashBufferIatroomtemperaturewithgentlerocking.Completelyremovewashbufferineachwashstep.Dilute20xWashBufferIwithH2O.
    -Decantthe1xWashBufferIfromeachwell,wash2times(5mineach)with150µlof1XWashBufferIIatroomtemperaturewithgentlerocking.Completelyremovewashbufferineachwashstep.Dilute20XWashBufferIIwithH2O.
    9.Reconstitutethedetectionantibodybyadding1.4mlofSampleDiluenttothetube.Spinbriefly.
    10.Add80µlofthedetectionantibodycocktailtoeachwell.Incubateatroomtemperaturefor1-2hour.
    11.Decantthesamplesfromeachwell,andwash5times(5minseach)with150µlof1XWashBufferIandthen2timeswith150µlof1xWashBufferIIatroomtemperaturewithgentlerocking.Completelyremovewashbufferineachwashstep.
    12.Afterbrieflyspinningdown,add1.4mlofSampleDiluenttoCy3equivalentdye-conjugatedstreptavidintube.Mixgently.
    13.Add80µlofCy3equivalentdye-conjugatedstreptavidintoeachwell.Coverthedevicewithaluminumfoiltoavoidexposuretolightorincubateindarkroom.Incubateatroomtemperaturefor1hour.
    14.Decantthesamplesfromeachwell,andwash5times(5minseach)with150µlof1XWashBufferIatroomtemperaturewithgentlerocking.Completelyremovewashbufferineachwashstep.
    15.Disassemblethedevicebypushingclipsoutwardfromtheslideside.Carefullyremovetheslidefromthegasket.
    16.PlacetheslideintheSlideWasher/Dryer(a4-slideholder/centrifugetube),addenough1xWashBufferI(about30ml)tocoverthewholeslide,andthengentlyshakeatroomtemperaturefor15minutes.DecantWashBufferI.Washwith1xWashBufferII(about30ml)andgentlyshakeatroomtemperaturefor5minutes.
    17.Removewaterdropletscompletelybygentlyapplyingsuctionwithapipettetoremovewaterdroplets.Donottouchthearray,onlythesides.
    18.Imaging:ThesignalscanbevisualizedthroughuseofalaserscannerequippedwithaCy3wavelength(greenchannel)suchasAxonGenePix.Makesurethatthesignalfromthewellcontainingthehigheststandardconcentration(Std1)receivesthehighestpossiblereading,yetremainsunsaturated.
CalculationofResults

DataextractioncanbedoneusingtheGALfilethatisspecificforthisarrayalongwiththemicroarrayanalysissoftware(GenePix,ScanArrayExpress,ArrayVision,MicroVigene,etc.).

AssayPrecisionReproducibility:CV< 20%
RestrictionsForResearchUseonly
HandlingAdviceDonottouchthesurfaceoftheslides,asthemicroarrayslidesareverysensitive.Holdtheslidesbytheedgesonly.Handleallbuffersandslideswithpowderfreegloves.Handleglassslide/sincleanenvironment.TheQuantibodyslidesdonothavebarcodes.Tohelpdistinguishoneslidefromanother,transcribetheslideserialnumberfromtheslidebagtothebackoftheslidewithanultra-finepointpermanentmarker.PleaseNote:Redpermanentmarkercansignificantlyinterferewithfluorescentsignaldetection.Werecommendmarkingyourslideswithagreen,blueorblackultra-finepointpermanentmarker.Pleasewritethenumberontheverybottomedgeoftheslide.Donotwriteonthearrayedwellareas.
Storage-20°C
StorageCommentForbestresults,storetheentirekitfrozenat-20°Cuponarrival.Storedfrozen,thekitwillbestableforatleast6monthswhichisthedurationoftheproductwarrantyperiod.Oncethawed,storearrayslide(s),standardmix,detectionantibodycocktail,andCy3-ConjugatedStreptavidinat-20°Candallotherreagentsundilutedat4°Cfornomorethan3months.
ExpiryDate6months
SupplierImages
image for Human Th1/Th4 Array Q1 (ABIN625754)HumanTh1/Th4ArrayQ1
Productcitedin:Schwarz,Klaus,Tudor,Fleischmann,Wekerle,Roth,Bodingbauer,Kaczirek:"TransectionSpeedandImpactonPerioperativeInflammatoryResponse-ARandomizedControlledTrialComparingStaplerHepatectomyandCUSAResection."in:PLoSONE,Vol.10,Issue10,pp.e0140314,2015(PubMed).

Epanchintsev,Shyamsunder,Verma,Lyakhovich:"IL-6,IL-8,MMP-2,MMP-9areoverexpressedinFanconianemiacellsthroughaNF-κB/TNF-αdependentmechanism."in:Molecularcarcinogenesis,Vol.54,Issue12,pp.1686-99,2015(PubMed).

Chia,Kumari,Casey:"BreastcancercellinvasionmediatedbyG?12signalinginvolvesexpressionofinterleukins-6and-8,andmatrixmetalloproteinase-2."in:Journalofmolecularsignaling,Vol.9,pp.6,2014(PubMed).

Green,Michno,Greenman,Stafford:"Effectoftreatmentonsystemiccytokinesinheadandnecksquamouscellcarcinomapatients."in:Resultsinimmunology,Vol.2,pp.1-6,2013(PubMed).

Knotkova,Rosedale,Strauss,Horne,Soto,Cruciani,Malaspina,Malamud:"UsingTranscranialDirectCurrentStimulationtoTreatDepressioninHIV-InfectedPersons:TheOutcomesofaFeasibilityStudy."in:Frontiersinpsychiatry,Vol.3,pp.59,2012(PubMed).

Wyatte,Herd,Mingus,OReilly:"TheRoleofCompetitiveInhibitionandTop-DownFeedbackinBindingduringObjectRecognition."in:Frontiersinpsychology,Vol.3,pp.182,2012(PubMed).

Stechova,Halbhuber,Hubackova,Kayserova,Petruzelkova,Vcelakova,Kolouskova,Ulmannova,Faresjö,Neuwirth,Spisek,Sediva,Filipp,Sumnik:"Casereport:type1diabetesinmonozygoticquadruplets."in:Europeanjournalofhumangenetics:EJHG,Vol.20,Issue4,pp.457-62,2012(PubMed).

Gao,Cheung,Li,Jiang,Chu,Duan,Cheung,Zhao,Choi,Dong,Tsim:"AChineseherbaldecoction,DangguiBuxueTang,activatesextracellularsignal-regulatedkinaseinculturedT-lymphocytes."in:FEBSletters,Vol.581,Issue26,pp.5087-93,2007(PubMed).

Kisliuk,Gaumont,Lafer,Baugh,Montgomery:"PolyglutamylderivativesoftetrahydrofolateassubstratesforLactobacilluscaseithymidylatesynthase."in:Biochemistry,Vol.20,Issue4,pp.929-34,1981(PubMed).


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