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2024年12月27日发(作者：之梦古钱币模板)
RatIL-1betaELISAKit
CatalogNO.:RK00009
version:2.0
Thispackageinsertmustbereadinitsentiretybefore
usingthisproduct
1
Introduction
Thekitisasandwichenzymeimmunoassayforinvitro
quantitativemeasurementofIL-1betainratserum,plasma,cell
culturesupernatants.
PrincipleoftheAssay
Thisassayemploysthequantitativesandwichenzymeimmunoassay
bodyspecificforratIL-1betahasbeen
rdsandsamplesarepipetted
intothewellsandanyIL-1betapresentisboundbythe
ashingawayanyunboundsubstances,
andthenadetectionantibodyspecificforIL-1betaisadded
tothewellsandbindstothecombinationofcaptureantibody
ingawashtoremoveanyunbound
combination,andenzymeconjugateisaddedtothewells.
Followingincubationandwashsteps,asubstratesolutionis
addedtothewellsandcolordevelopsinproportiontotheamount
ordevelopment
isstoppedandtheabsorbanceismeasured.
2
MaterialProvided&StorageConditions
Unopenedkitscanbestoredat2-8°Cfor1year,andopened
productsmustbeusedwithin1month.
Storageof
/reconstituted
material
Puttheunusedslatsback
inthealuminumfoilbag
AntibodyCoated
Plate
8×12
RM00064
withthedesiccantand
nbe
storedat2-8°Cfor1
month.
Standard
Lyophilized
Itisnotrecommendedto
2vials
RM00061
useagainafter
redissolving.
Concentrated
Biotin
Conjugate
Antibody(100
×)
1×120ulRM00062
Storeat2-8°cfor1month
*
3
Streptavidin-HR
PConcentrated
(100×)
Standard/Sample
Diluent(R1)
Biotin-Conjugat
eAntibody
Diluent(R2)
Streptavidin-HR
PDiluent(R3)
Wash
Buffer(20x)
TMBSubstrate
StopSolution
PlateSealers
Specification
1×12mLRM00025
Storeat2-8°cfor1month
*
1×30mL
1×12mL
1×6mL
4Strips
1
RM00026
RM00027
RM00028
1×12mLRM00024
1×20mLRM00023
1×120ulRM00063
Storeat2-8°cfor1month
*
4
OtherSuppliesRequired
latereadercapableofmeasuringabsorbanceat
450nm,withthecorrectionwavelengthsetat630nmor570
nm.
2.
3.
4.
Pipettesandpipettetips.
Deionizedordistilledwater.
Squirtbottle,manifolddispenser,orautomatedmicroplate
washer.
5.
6.
Incubator.
Testtubesfordilutionofstandardsandsamples.
5
Precautions
iationindiluent,operator,pipettingtechnique,
washingtechnique,incubationtimeortemperature,andkit
agecancausevariationinbinding.
ionsinsamplecollection,processing,andstorage
maycausesamplevaluedifferences.
tsmaybeharmful,ifingested,rinseitwithan
excessamountoftapwater.
e,hand,and
faceprotection.
performsimplecentrifugationtocollecttheliquid
beforeuse.
ixorsubstitutereagentswiththosefromotherlots
orothersources.
temixingisparticularlyimportantforgoodresult.
Useamini-vortexeratthelowestfrequency.
sampleandallcomponentsinthekitsadequately,
andusecleanplasticcontainertopreparealldiluents.
esampleandstandardshouldbeassayedinduplicate,
6
andreagentsshouldbeaddedinsequenceinaccordancewith
therequirementofthespecification.
10.
11.
Reuseofdissolvedstandardisnotrecommended.
Thekitshouldnotbeusedbeyondtheexpirationdateon
thekitlabel.
shouldbeawayfromlightwhenitisstoredor
incubated.
cethelikelihoodofblood-bornetransmissionof
infectiousagents,handleallserum,plasma,andother
biologicalfluidsinaccordancewithNCCLSregulations.
dcrosscontamination,pleaseusedisposable
pipettetips.
prepareallthekitcomponentsaccordingtothe
itswillbeusedseveraltimes,
pleasesealthereststripsandpreservewithdesiccants.
Douseupwithin2months.
sayisdesignedtoeliminateinterferencebyother
factorspresentinbiologicalsamples.
llfactorshavebeentestedinthisassay,the
possibilityofinterferencecannotbeexcluded.
48Tkitisalsosuitableforthespecification.
7
SampleCollection&Storage
Thesamplecollectionandstorageconditionslistedbeloware
stabilityhasnotbeen
evaluated.
SamplescontainingthecorrelatedIgGasinthiskitmay
interferewiththisassay.
CellCultureSupernatant:Removeparticulatesbycentrifugation.
Assayimmediatelyoraliquotandstoresamplesat≤-20°C.
Avoidrepeatedfreeze-thawcycles.
Serum:Useaserumseparatortube(SST)andallowsamplestoclot
for30minutesatroomtemperaturebeforecentrifugationfor15
serumandassayimmediatelyoraliquot
andstoresamplesat≤-20°epeatedfreeze-thaw
cycles.
Plasma:CollectplasmausingEDTAorHeparinasananticoagulant.
Centrifugefor15minutesat1000×gwithin30minutesof
mmediatelyoraliquotandstoresamplesat
≤-20℃.Avoidrepeatedfreeze-thawcycles.(Note:Citrate
8
plasmahasnotbeenvalidatedforuseinthisassay.)
Note:Itissuggestedthatallsamplesinoneexperimentbe
emolyticand
hyperlipidemiasampleforserumandplasma.
ReagentPreparation
tals
haveformedintheconcentrate,Bringthereagenttoroom
temperatureandmixgentlyuntilthecrystalshavecompletely
dissolved.
Standard-ReconstitutetheStandardLyophilizedwith1.0mL
Standard/SampleDiluent(R1).Thisreconstitutionproducesa
stocksolutionof8000pg/standardtoensurecomplete
reconstitutionandallowthestandardtositforaminimumof
15minuteswithgentleagitationpriortomakingdilutions.
Usethe8000pg/mLstandardstocktoproduceadilutionseries
(below)withStandard/SampleDiluent(R1).Mixeachtube
thoroughlyandchangepipettetipsbetweeneachtransfer
9
(recommendedconcentrationforstandardcurve:4000,2000,1000,
Std250μL
500,250,125,62.5,0pg/mL).Usedilutedstandardswithin60
250μL250μL250μL250μL
250μL250μL
minutesofpreparation.
R11000μL
250μL
250μL250μL
250μL
250μL
250μL
250μL
250μL
8000pg/mL
4000pg/mL
2000pg/mL1000pg/mL
500pg/mL
250pg/mL
125pg/mL62.5pg/mL
0pg/mL
WorkingBiotinConjugateAntibody-Dilute1:100ofConcentrated
BiotinConjugateAntibody(100x)withBiotin-ConjugateAntibody
Diluent(R2)beforeuse,forexample:Add20μLofConcentrated
BiotinConjugateAntibody(100x)to1980μLBiotin-Conjugate
AntibodyDiluent(R2)toprepare2000μLWorkingBiotin
ConjugateAntibodyBuffer.
WorkingStreptavidin-HRP-Dilute1:100ofConcentrated
Streptavidin-HRP(100x)withStreptavidin-HRPDiluent(R3)
beforeuse,forexample:Add20μLofConcentrated
Streptavidin-HRP(100x)to1980μLStreptavidin-HRPDiluent(R3)
10
toprepare2000μLWorkingStreptavidin-HRPBuffer.
WashBuffer-Ifcrystalshaveformedintheconcentrate,warm
toroomtemperatureandmixgentlyuntilthecrystalshave
1:20withdoubledistilledor
deionizedwaterbeforeuse,forexample:Add20mLofWashBuffer
Concentrateto380mLofdeionizedordistilledwatertoprepare
400mLofWashBuffer.
11
AssayProcedure
Bringallreagentsandsamplestoroomtemperaturebeforeuse.
Itisrecommendedthatallstandards,controls,andsamplesbe
assayedinduplicate.
eallreagents,workingstandards,andsamplesas
directedintheprevioussections.
excessmicroplatestripsfromtheplateframe,
returnthemtothefoilpouchcontainingthedesiccantpack,
andresealproperly.
hbuffer350μL/well,aspirateeachwellafter
holding40seconds,repeatingtheprocesstwotimesfor
atotalofthreewashes.
4.
5.
Add100μLStandard/sampleDiluent(R1)inablankwell.
Add100μLdifferentconcentrationofstandardorsample
inotherwells,Coverwiththeadhesivestripprovided.
Incubatefor2hoursat37℃.recordtheplatelayoutof
standardsandsampleassay.
etheConcentratedBiotinConjugateAntibody(100x)
WorkingSolution15minutesearlybeforeuse.
7.
8.
Repeattheaspiration/washasinstep3.
Add100μLWorkingBiotinConjugateAntibodyineachwell,
tefor1
12
hourat37℃.
etheStreptavidin-HRPConcentrated(100x)Working
Solution15minutesearlybeforeuse.
10.
11.
Repeattheaspiration/washasinstep3.
Add100μLWorkingStreptavidin-HRPineachwell,cover
tefor0.5hour
at37℃.
12.
13.
Repeattheaspiration/washasinstep3.
Duringtheincubation,turnonthemicroplatereaderto
warmupfor30minutesbeforemeasuring.
100μtefor15-20
minutesat37℃.Protectfromlight.
50μLStopSolution,determinetheopticaldensityof
eachwellwithin5minutes,usingaMicroplatereaderset
lengthcorrectionisavailable,setto
lengthcorrectionisnotavailable,
subtractreadingsat570nmor630nmfromthereadings
btractionwillcorrectforoptical
gsmadedirectlyat450
nmwithoutcorrectionmaycausehighervalueandless
accurateresult.
13
AssayProcedureSummary
Preparethestandardandreagents
Wash3times
↓
Add100ulofstandardsortestsamplestoeachwell
Incubatefor2hoursat37℃,thenwash3times
↓
Add100ulWorkingBiotinConjugateAntibody
Incubatefor1hourat37℃,thenwash3times
↓
Add100ulWorkingStreptavidin-HRP
Incubatefor0.5hourat37℃,thenwash3times
↓
Add100ulSubstrateSolution
Incubatefor15-20minat37℃underdarkcondition
↓
Add50ulStopSolution
↓
Detecttheopticaldensitywithin5minutesunder450nm.
CorrectionWavelengthsetat570nmor630nm
14
CalculationofResults
etheduplicatereadingsforeachstandard,control
andsample,andsubtracttheaveragezerostandardoptical
density(O.D.).
astandardcurvebyreducingthedatausingcomputer
softwarecapableofgeneratingafour-parameterlogistic
(4-PL)ternative,constructastandard
curvebyplottingthemeanabsorbanceforeachstandard
ontheY-axisagainsttheconcentrationontheX-axisand
drawabestfitcurvethroughthepointsonalog/loggraph.
ThedatamaybelinearizedbyplottingthelogoftheIL-1
ear
scale,andthebestfitlinecanbedeterminedby
regressionanalysis.
leshavebeendiluted,theconcentrationreadfrom
thestandardcurvemustbemultipliedbythedilution
factor.
TypicalData
15
Thestandardcurvesareprovidedfordemonstrationonly.A
standardcurveshouldbegeneratedforeachsetofIL-1beta
assayed.
DetectionRange
62.5-4000pg/mL
Sensitivity
16
Theminimumdetectabledose(MDD)ofIL-1betatypicallyless
than13.22pg/wasdeterminedbyaddingtwostandard
deviationstothemeanopticaldensityvalueoftwentyzero
standardreplicatesandcalculatingthecorresponding
concentration.
Specificity
17
ThisassayrecognizesbothrecombinantandnaturalratIL-1beta.
Thefactorslistedbelowwerepreparedat50ng/mlandassayed
ificantcross-reactivitywas
observedwiththefollowing:
Recombinantrat:
IL-10
IFN-γ
IL-1α
IL-2
IL-4
IL-6
IL-8
TNF-α
Recombinanthuman:
IL-1
IL-2
IL-6
Note:
Limitedbycurrentskillsandknowledge,itisimpossiblefor
ustocompletethecross-reactivitydetectionbetweenIL-1beta
andalltheanalogues,therefore,crossreactionmaystillexist.
18
Precision
Intra-platePrecision
3sampleswithlow,middleandhighlevelIL-1betaweretested
20timesononeplate,respectively.
Intra-Assay:CV<10%
Inter-platePrecision
3sampleswithlow,middleandhighlevelIL-1betaweretested
on3differentplates,20replicatesineachplate.
Inter-Assay:CV<15%
Intra-AssayPrecision
Sample
n
Mean(pg/mL)
Standarddeviation
CV(%)
1
20
1165
47.8
4.1
2
20
2268
79.4
3.5
3
20
3535
137.9
3.9
Inter-AssayPrecision
1
20
1160
75.4
6.5
2
20
2259
162.6
7.2
3
20
3542
219
6.2
19
Recovery
MatriceslistedbelowwerespikedwithcertainlevelofIL-1beta
andtherecoveryrateswerecalculatedbycomparingthemeasured
valuetotheexpectedamountofIL-1betainsamples.
Sample
CellCultureMedia(n=5)
Serum(n=5)
AverageRecovery（%）
104
99
Range（%）
95-113
90-108
20
Linearity
Thelinearityofthekitwasassayedbytestingsamplesspiked
withappropriateconcentrationofIL-1betaandtheirserial
ultsweredemonstratedbythepercentageof
calculatedconcentrationtotheexpected.
CellCultureMedia(n=5)
103
98-108
98
84-111
96
Serum(n=5)
95
87-102
102
89-114
100
//
AverageofExpected（%）
1:2
Range（%）
AverageofExpected（%）
1:4
Range（%）
AverageofExpected（%）
1:8
Range（%）86-10692-108
AverageofExpected（%）
1:16
Range（%）
98
91-105
106
96-115
21
TroubleShooting
ProblemPossibleCauseSolution
Sufficientlywashplatesas
appropriate
Insufficientwashingdurationandnumberofwashes.
Ensureappropriatevolumeofwash
bufferineachwell.
High
Background
Incorrectincubation
procedure
Checkwhetherthedurationand
temperatureofincubationaresetup
asrequired.
Becarefuloftheoperationsthat
Cross-contaminationof
samplesandreagents
couldcausecross-contamination.
Usefreshreagentsandrepeatthe
tests.
Checktheconcentrationand
Incorrectuseof
reagents
suretousereagentsinproper
order.
Nosignalor
weaksignal
Incorrectuseof
microplatereader
suretosetupappropriatemain
wavelengthandcorrection
wavelength.
Insufficientcolour
reactiontime
Optimumdurationofcolourreaction
shouldbelimitedto15-25minutes.
22
Readtoolateafter
stoppingthecolour
reaction
Matrixeffectof
samples
ContaminationofTMB
substrate
Toomuch
signal
Platesealersreused
Proteinconcentration
insampleistoohigh
Unevenadditionof
samples
Impuritiesand
Poor
Duplicates
precipitatesin
samples
Inadequatemixingof
reagents
Readtheplatein5minutesafter
stoppingthereaction.
Usepositivecontrol.
CheckifTMBsubstratesolution
TMBsubstrate
solution.
Useafreshnewsealerineachstep
ofexperiments.
Dopre-testanddilutesamplesin
optimumdilutionratio.
ically
calibratethepipette.
Centrifugesamplesbeforeuse.
Mixallsamplesandreagentswell
beforeloading.
*therapeuticordiagnostic
purposes.
23
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