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Page1of4DocumentNo:IFU.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISARatandMouseAMHELISAAL-113INTENDEDUSETheRatandMouseAnti-Müllerianhormone(AMH)enzyme-linkedimmunosorbentassay(ELISA)kitprovidesmaterialsforthequantitativemeasurementofAMHinratandmouseserumandotherbiologicalfluids.
SUMMARYANDEXPLANATIONAnti-Müllerianhormoneisa140kDaglycoproteinthatisproducedduringnormalembryogenesisbytheSertolicellsoftheembryonictestis,causestheinvolutionoftheMüllerianduct,andinhibitsfemalegonadogenesisbyinducingapoptosisoftargetgonadalcells.
Itbelongstothetransforminggrowthfactor-βsuperfamily.
AMHcausesapoptosisofspecificAnti-Müllerianinhibitingsubstance(MIS)receptor-bearingcells,whilehavingnoeffectoncellswithoutreceptors.
AMHisalsoexpressedingranulosacellsofpreantralandsmallantralfolliclesintheovary,andAMHinhibitsrecruitmentofprimordialfolliclesintothepoolofgrowingfollicles,anddecreasesresponsivenessofgrowingfolliclestoFSH.
PRINCIPLEOFTHETESTTheRatandMouseAMHELISAisaquantitativethree-stepsandwichtypeimmunoassay.
InthefirststepseriallydilutedCalibratorsandunknownsamplesareaddedtoAMHantibodycoatedmicrotiterwellsandincubated.
Afterthefirstincubationandwashing,thewellsareincubatedwithbiotinylatedAMHantibodysolution.
Aftersecondincubationandwashing,thewellsareincubatedwithstreptavidinhorseradishperoxidaseconjugate(SHRP)solution.
Afterthethirdincubationandwashingstep,thewellsareincubatedwithsubstratesolution(TMB)followedbyanacidicstoppingsolution.
Inprinciple,theantibody-biotinconjugatebindstothesolidphaseantibody-antigencomplexwhichinturnbindstothestreptavidin-enzymeconjugate.
Theantibody-antigen-biotinconjugate-SHRPcomplexboundtothewellisdetectedbyenzyme-substratereaction.
Thedegreeofenzymaticturnoverofthesubstrateisdeterminedbydualwavelengthabsorbancemeasurementat450nmasprimarytestfilterand630nmasreferencefilter.
TheabsorbancemeasuredisdirectlyproportionaltotheconcentrationofAMHinthesamplesandcalibrators.
MATERIALSSUPPLIEDCAL-105AAMH/MISCalibratorsA/SampleDiluentOnebottle,11mL,labeledAMH/MISCalA/SampleDiluent,containing0ng/mLAMHinproteinbasedbufferandPro-Clean400.
Storeunopenedat2-8°Cuntiltheexpirationdate.
CAL-113HRatandMouseAMHCalibratorH(Lyophilized)ReconstitutetheRatandMouseAMHCalibratorHwith1mLofdeionizedwater.
Solubilize,mixwellanduseafterreconstitution.
TheconcentrationofthecalibratorHinthestocksolutionisapproximately20ng/mL.
Refertotheviallabelforexactconcentration.
Storeunopenedat2-8°Cuntiltheexpirationdate.
PLT-113AMHCoatedMicrotitrationstripsOnestripholder,containing12stripsand96microtitrationwellswithAMHantibodyimmobilizedtotheinsidewallofeachwell.
Storeat2-8°Cuntilexpirationdateintheresealablepouchwithadesiccanttoprotectfrommoisture.
ASB-113AMHAssayBufferOnebottle,8mL,containingaprotein-based(BSA)-bufferwithanon-mercurypreservative.
Storeat2-8°Cuntilexpirationdate.
BCR-113AMHBiotinConjugateReady-To-Use(RTU)Onebottle,12mL,containingbiotinylatedanti-AMHantibodyinprotein-basedbufferwithanon-mercurypreservative.
Storeat2-8°Cuntilexpirationdate.
SAR-113AMHStreptavidin-EnzymeConjugate-Ready-to-Use(RTU)Onebottle,12mL,containingstreptavidin-HRP(horseradishperoxidase)inaprotein-basedbufferandanon-mercurypreservative.
Storeundilutedat2-8Cuntilexpirationdate.
TMB-100TMBChromogenSolutionOnebottle,12mL,containingasolutionoftetramethylbenzidine(TMB)inbufferwithhydrogenperoxide.
Storeat2-8°Cuntilexpirationdate.
STP-100StoppingSolutionOnebottle,12mL,containing0.
2Msulfuricacid.
Storeat2to30°Cuntilexpirationdate.
WSH-100WashConcentrateAOnebottle,60mL,containingbufferedsalinewithanonionicdetergent.
Storeat2-30Cuntilexpirationdate.
Dilute25-foldwithdeionizedwaterpriortouse.
MATERIALSREQUIREDBUTNOTPROVIDED1.
Microtitrationplatereadercapableofabsorbancemeasurementat450nm,405nmand630nm.
2.
Microplateshaker.
3.
Microplatewasher.
4.
Semi-automated/manualprecisionpipettetodeliver10–250μL.
5.
Repeaterpipette.
6.
Vortexmixer.
7.
Deionizedwater.
WARNINGSANDPRECAUTIONSForin-vitroresearchuse.
Thefollowingprecautionsshouldbeobserved:a)Followgoodlaboratorypractice.
b)Usepersonalprotectiveequipment.
Wearlabcoatsanddisposablegloveswhenhandlingimmunoassaymaterials.
Page2of4DocumentNo:IFU.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISAc)HandleanddisposeofallreagentsandmaterialincompliancewithapplicableregulationsWARNING:PotentialBiohazardousMaterialThisreagentmaycontainsomeanimaland/orhumansourcematerial(e.
g.
serum)ormaterialsusedinconjunctionwithhumansourcematerials.
HandleallreagentsandpatientsamplesataBiosafetyLevel2,asrecommendedforanypotentiallyinfectioushumanmaterialintheCentersforDiseaseControl/NationalInstitutesofHealthmanual"BiosafetyinMicrobiologicalandBiomedicalLaboratories,"5thEdition,20071.
WARNING:PotentialChemicalHazardSomereagentsinthiskitcontainPro-Clean400andSodiumazide2asapreservative.
Pro-Clean400andSodiumAzideinconcentratedamountsareirritantstoskinandmucousmembranes.
Forfurtherinformationregardinghazardoussubstancesinthekit,pleaserefertotheMSDS,eitheratAnshLabs.
comorbyrequest.
SAMPLECOLLECTIONa)Serumistherecommendedsampletype.
b)Samplehandling,processing,andstoragerequirementsdependonthebrandofbloodcollectiontubethatyouuse.
Pleasereferencethemanufacturer'sinstructionsforguidance.
Eachlaboratoryshoulddeterminetheacceptabilityofitsownbloodcollectiontubesandserumseparationproducts.
c)Samplesmaybestoredat4°Cifassayedwithin24hours;otherwisesamplesmustbestoredat-20°Cor-80°Ctoavoidlossofbioactivityandcontamination.
d)Avoidassayinglipemic,hemolyzedorictericsamples.
e)Avoidrepeatedfreezingandthawingofsamples.
Thawsamplesnomorethan3times.
f)Forshipping,placespecimensinleakproofcontainersinbiohazardspecimenbagswithappropriatespecimenidentificationandtestrequisitioninformationintheoutsidepocketofthebiohazardspecimenbag.
FollowDOTandIATArequirementswhenshippingspecimens.
PROCEDURALNOTES1.
AthoroughunderstandingofthispackageinsertisnecessaryforsuccessfuluseoftheRatandMouseAMHELISAassay.
Itisthelaboratory'sresponsibilitytovalidatetheassayfortheiruse.
Accurateresultswillonlybeobtainedbyusingpreciselaboratorytechniquesandfollowingthepackageinsert.
2.
Acalibrationcurvemustbeincludedwitheachassay.
3.
Bringallkitreagentstoroomtemperaturebeforeuse.
Thoroughlymixthereagentsbeforeusebygentleinversion.
Donotmixvariouslotsofanykitcomponentanddonotuseanycomponentbeyondtheexpirationdate.
4.
Useacleandisposablepipettetipforeachreagent,calibrator,controlorsample.
Avoidmicrobialcontaminationofreagents,contaminationofthesubstratesolutionswiththeHRPconjugates.
Theenzymeusedasthelabelisinactivatedbyoxygen,andishighlysensitivetomicrobialcontamination,sodiumazide,hypochlorousacidandaromaticchlorohydrocarbonsoftenfoundinlaboratorywatersupplies.
Usedeionizedwater.
5.
Incompletewashingwilladverselyaffecttheoutcomeandassayprecision.
CareshouldbetakentoaddTMBintothewellstominimizepotentialassaydriftduetovariationintheTMBincubationtime.
Avoidexposureofthereagentstoexcessiveheatordirectsunlight.
PREPARATIONOFREAGENTS1.
AMHCalibratorsH:a.
TapandreconstituteRatandMouseAMHCalibratorHwith1mLdeionizedwater.
Solubilizefortenminutes,mixwellbeforeuse.
b.
PreparesevenpolystyrenetubesandlabelthemasCalA,CalB,CalC,CalD,CalE,CalFandCalG.
c.
Add500lofAMH/MISCalibratorA/SampleDiluenttoeachpolystyrenetubelabeledCalA-G.
d.
Add500lofreconstitutedAMHCalibratorH(fromstepa)tothetubelabeledCalG.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
e.
Add500lofCalG(fromstepd)tothetubelabeledCalF.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
f.
Add500lofCalF(fromstepe)tothetubelabeledCalE.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
g.
Add500lofCalE(fromstepf)tothetubelabeledCalD.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
h.
Add500lofCalD(fromstepg)tothetubelabeledCalC.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
i.
Add500lofCalC(fromsteph)tothetubelabeledCalB.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
j.
ThetubelabeledCalAcontains500lAMHCalibratorA/SampleDiluentandhas0AMHconcentrationsandshouldbeusedasBlank.
NOTE:Ifgreatersensitivityisdesired,CalBcanbefurtherdilutedtoB/2usingCalA.
k.
TheCalibratorsA-Hforinstanceshouldreadas0.
0ng/mL,0.
313ng/mL,0.
625ng/mL,1.
25ng/mL,2.
5ng/mL,5ng/mL,10ng/mLand20ng/mL.
AliquotandfreezetheRatandMouseAMHCalHStockimmediatelyformultipleuses.
Avoidrepeatedfreezethaws.
Frozenaliquotsat-20°Caregoodforoneyear.
l.
TheAMHconcentrationintheRatandMouseAMHcalibratorsHistraceabletothemanufacturer'sworkingcalibrators.
Valuesassignedbyothermethodologiesmaybedifferent.
Suchdifferences,ifpresent,maybecausedbyinter-methodbias.
2.
WashSolution:Dilutewashconcentrate25-foldwithdeionizedwater.
Thewashsolutionisstableforonemonthatroomtemperaturewhenstoredinatightlysealedbottle.
3.
MicrotitrationWells:Selectthenumberofcoatedwellsrequiredfortheassay.
Theremainingunusedwellsshouldbeplacedintheresealablepouchwithadesiccant.
Thepouchmustberesealedtoprotectfrommoisture.
SAMPLEPREPARATION1.
Maleratandmousesamples:Dilute1:50intoCalibratorA/SampleDiluent.
(1partunknownsampleinto49partsCalibratorA/SampleDiluent).
2.
Femaleratandmousesamples:Dilute1:10intoCalibratorA/SampleDiluent.
(1partunknownsampleinto9partsCalibratorA/SampleDiluent).
Page3of4DocumentNo:IFU.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISANOTE:AMHvaluescanvaryfromonebreedtoanother.
Youmayneedtoestablishyourdilutionfactoraccordinglytoensurethedilutedsamplesfallwithinthemeasurablerangeoftheassay.
ASSAYPROCEDUREAllowallspecimensandreagentstoreachroomtemperature(23±2C)andmixthoroughlybygentleinversionbeforeuse.
NOTE:Allsamplesshouldbedilutedasperthe"SamplePreparation"section.
NOTE:Calibratorsanddilutedunknownsshouldbeassayedinduplicate.
NOTE:Alldilutedsamplesreadinghigherthanthehighestcalibratorshouldbefurtherdilutedinthe0ng/mLCalibratorA/Samplediluentpriortoassay.
1.
Labelthemicrotitrationstripstobeused.
2.
Pipette50μLoftheCalibrators(CalA-H)anddilutedunknowns(See"SamplePreparation"section)totheappropriatewells.
3.
Add50μLoftheAMHAssayBuffertoeachwellusingarepeaterpipette.
4.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for120minutesatroomtemperature(23±2C).
5.
Aspirateandwasheachstrip5timeswithWashSolution(350uL/well)usinganautomaticmicroplatewasher.
6.
Add100μLoftheAntibody-BiotinConjugateRTUtoeachwellusingarepeaterpipette.
7.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for60minutesatroomtemperature(23±2C).
8.
Aspirateandwasheachstrip5timeswiththeWashSolution(350uL/well)usinganautomaticmicroplatewasher.
9.
Add100LoftheStreptavidin-EnzymeConjugate-RTUtoeachwellusingarepeaterpipette.
10.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for30minutesatroomtemperature(23±2C).
11.
Aspirateandwasheachstrip5timeswiththeWashSolution(350uL/well)usinganautomaticmicroplatewasher.
12.
Add100μLoftheTMBchromogensolutiontoeachwellusingarepeaterpipette.
Avoidexposuretodirectsunlight.
13.
Incubatethewells,shakingat600–800rpmonanorbitalmicroplateshaker,for8-12minatroomtemperature(23±2°C).
NOTE:Visuallymonitorthecolordevelopmenttooptimizetheincubationtime.
14.
Add100μLoftheStoppingsolutiontoeachwellusingarepeaterpipette.
Readtheabsorbanceofthesolutioninthewellswithin20minutes,usingamicroplatereadersetto450nm.
NOTE:Zerocalibratorshouldbeprogrammedas"Blank"whilereadingtheopticaldensity.
Ifinstrumenthasawavelengthcorrection,settheinstrumenttodualwavelengthmeasurementat450nmwithbackgroundwavelengthcorrectionat630nm.
RESULTSNOTE:Theresultsinthispackageinsertwerecalculatedbyplottingthelogopticaldensity(OD)dataonthey-axisandlogAMHconcentrationonX-axisusingacubicregressioncurve-fit.
Alternatively,logvs.
logquadraticregressioncurve-fitcanbeused.
Otherdatareductionmethodsmaygiveslightlydifferentresults.
1.
Optimumresultscanbeobtainedatincubationtemperatureof23±2°C.
2.
Calculatethemeanopticaldensity(OD)foreachcalibrator,Control,orUnknown.
3.
PlotthelogofthemeanODreadingsforeachoftheCalibratorsalongthey-axisversuslogoftheAMHconcentrationsinng/mLalongthex-axis,usingacubicregressioncurve-fit.
4.
DeterminetheAMHconcentrationsoftheunknownsfromthecalibrationcurvebymatchingtheirmeanODreadingswiththecorrespondingAMHconcentrations.
5.
AnysamplereadinghigherthanthehighestCalibratorshouldbeappropriatelydilutedwiththe0ng/mL(CALA/SampleDiluent)andre-assayed.
6.
Anysamplereadinglowerthantheanalyticalsensitivityshouldbereportedassuch.
7.
Multiplytherecoveredunknownvaluesbytheappropriatedilutionfactor.
LIMITATIONSThereagentssuppliedinthiskitareoptimizedtomeasureAMHlevelsinratandmouseserum.
Ifthereisevidenceofmicrobialcontaminationorexcessiveturbidityinareagent,discardthevial.
Forassaysemployingantibodies,thepossibilityexistsforinterferencebyheterophileantibodiesinthesamples4.
QUALITYCONTROLEachlaboratoryshouldestablishmeanvaluesandacceptablerangestoassureproperperformance.
EachlaboratoryshouldestablishinternalAMHcontrolsranges.
Theresultsshouldfallwithinestablishedconfidencelimits.
Afullcalibrationcurve,andcontrol,shouldbeincludedineachassay.
TMBshouldbecolorless.
Developmentofanycolormayindicatereagentcontaminationorinstability.
REPRESENTATIVECALIBRATIONCURVEDATAWellNumberWellContentsMeanAbsorbanceConc(ng/mL)A1,A2CalibratorsA0.
060(Blank)0B1,B2B0.
060.
336C1,C2C0.
1360.
672D1,D2D0.
2521.
344E1,E2E0.
5112.
687F1,F2F0.
9885.
375G1,G2G1.
93410.
75H1,H2H3.
41921.
5CAUTION:Theabovedatamustnotbeemployedinlieuofdataobtainedbytheuserinthelaboratory.
NOTE:TheODvalueslistedaboveforcalibratorsB-Hareblanksubtracted.
ANALYTICALCHARACTERISTICSAllanalyticalcharacteristicsarestatedinng/mL(1ng/mLAMH=7.
14pmol/L)Specificity:TheAnshLabsRatandMouseAMHassayhasbeenscreenedforcrossreactivitywithotherspeciesAMH,including:rabbit,avian,hamster,belugawhale,andbottlenosedolphin.
TheantibodiesexhibithighaffinitytoAMHofthesespecies.
TestsforparallelismbylinearlydilutingspecimensfromthesespeciesandassayingthedilutedserumdemonstratetheELISAkitaccuracyfordeterminingAMHintheseanimalsLinearity:MultipledilutionsofsamplescontainingvariousAMH/MISlevelsweredilutedwithCalibratorA/samplediluent.
The%recoveryonindividualsamplesisrepresentedinthefollowingtable.
SampleDilutionFactorExpectedConc.
(ng/mL)ObservedConc.
(ng/mL)%RecoveryHamster1:101:201:401:802.
21.
10.
550.
27NA1.
10.
680.
31NA100%124%115%Mouse11:201:401:8020.
66510.
3335.
166NA9.
8654.
771NA95%92%Page4of4DocumentNo:IFU.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISA1:1602.
5832.
40393%Mouse21:101:201:4021.
69610.
8485.
424NA11.
1715.
710NA103%105%Mouse31:101:201:4019.
7399.
8704.
935NA10.
3285.
206NA105%105%RecombinantRatAMHNeat1:021:041:089.
934.
972.
481.
24NA4.
782.
211.
04NA96%89%84%REFERENCES1.
HHSPublication,5thed.
,2007.
BiosafetyinMicrobiologicalandBiomedicalLaboratories.
Availablehttp://www.
cdc.
gov/biosafety/publications/bmbl5/BMBL52.
DHHS(NIOSH)PublicationNo.
78–127,August1976.
CurrentIntelligenceBulletin13-ExplosiveAzideHazard.
Availablehttp://www.
cdc.
gov/niosh.
3.
ApprovedGuideline–ProceduresfortheHandlingandProcessingofBloodSpecimens,H18-A3.
2004.
ClinicalandLaboratoryStandardsInstitute.
4.
KrickaL.
Interferencesinimmunoassays–stillathreat.
ClinChem2000;46:1037–1038.
TheAnshLabslogoisatrademarkofAnshLabs.
Manufacturedby:AnshLabs445MedicalCenterBlvd.
Webster,TX77598-4217,U.
S.
A.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISARatandMouseAMHELISAAL-113INTENDEDUSETheRatandMouseAnti-Müllerianhormone(AMH)enzyme-linkedimmunosorbentassay(ELISA)kitprovidesmaterialsforthequantitativemeasurementofAMHinratandmouseserumandotherbiologicalfluids.
SUMMARYANDEXPLANATIONAnti-Müllerianhormoneisa140kDaglycoproteinthatisproducedduringnormalembryogenesisbytheSertolicellsoftheembryonictestis,causestheinvolutionoftheMüllerianduct,andinhibitsfemalegonadogenesisbyinducingapoptosisoftargetgonadalcells.
Itbelongstothetransforminggrowthfactor-βsuperfamily.
AMHcausesapoptosisofspecificAnti-Müllerianinhibitingsubstance(MIS)receptor-bearingcells,whilehavingnoeffectoncellswithoutreceptors.
AMHisalsoexpressedingranulosacellsofpreantralandsmallantralfolliclesintheovary,andAMHinhibitsrecruitmentofprimordialfolliclesintothepoolofgrowingfollicles,anddecreasesresponsivenessofgrowingfolliclestoFSH.
PRINCIPLEOFTHETESTTheRatandMouseAMHELISAisaquantitativethree-stepsandwichtypeimmunoassay.
InthefirststepseriallydilutedCalibratorsandunknownsamplesareaddedtoAMHantibodycoatedmicrotiterwellsandincubated.
Afterthefirstincubationandwashing,thewellsareincubatedwithbiotinylatedAMHantibodysolution.
Aftersecondincubationandwashing,thewellsareincubatedwithstreptavidinhorseradishperoxidaseconjugate(SHRP)solution.
Afterthethirdincubationandwashingstep,thewellsareincubatedwithsubstratesolution(TMB)followedbyanacidicstoppingsolution.
Inprinciple,theantibody-biotinconjugatebindstothesolidphaseantibody-antigencomplexwhichinturnbindstothestreptavidin-enzymeconjugate.
Theantibody-antigen-biotinconjugate-SHRPcomplexboundtothewellisdetectedbyenzyme-substratereaction.
Thedegreeofenzymaticturnoverofthesubstrateisdeterminedbydualwavelengthabsorbancemeasurementat450nmasprimarytestfilterand630nmasreferencefilter.
TheabsorbancemeasuredisdirectlyproportionaltotheconcentrationofAMHinthesamplesandcalibrators.
MATERIALSSUPPLIEDCAL-105AAMH/MISCalibratorsA/SampleDiluentOnebottle,11mL,labeledAMH/MISCalA/SampleDiluent,containing0ng/mLAMHinproteinbasedbufferandPro-Clean400.
Storeunopenedat2-8°Cuntiltheexpirationdate.
CAL-113HRatandMouseAMHCalibratorH(Lyophilized)ReconstitutetheRatandMouseAMHCalibratorHwith1mLofdeionizedwater.
Solubilize,mixwellanduseafterreconstitution.
TheconcentrationofthecalibratorHinthestocksolutionisapproximately20ng/mL.
Refertotheviallabelforexactconcentration.
Storeunopenedat2-8°Cuntiltheexpirationdate.
PLT-113AMHCoatedMicrotitrationstripsOnestripholder,containing12stripsand96microtitrationwellswithAMHantibodyimmobilizedtotheinsidewallofeachwell.
Storeat2-8°Cuntilexpirationdateintheresealablepouchwithadesiccanttoprotectfrommoisture.
ASB-113AMHAssayBufferOnebottle,8mL,containingaprotein-based(BSA)-bufferwithanon-mercurypreservative.
Storeat2-8°Cuntilexpirationdate.
BCR-113AMHBiotinConjugateReady-To-Use(RTU)Onebottle,12mL,containingbiotinylatedanti-AMHantibodyinprotein-basedbufferwithanon-mercurypreservative.
Storeat2-8°Cuntilexpirationdate.
SAR-113AMHStreptavidin-EnzymeConjugate-Ready-to-Use(RTU)Onebottle,12mL,containingstreptavidin-HRP(horseradishperoxidase)inaprotein-basedbufferandanon-mercurypreservative.
Storeundilutedat2-8Cuntilexpirationdate.
TMB-100TMBChromogenSolutionOnebottle,12mL,containingasolutionoftetramethylbenzidine(TMB)inbufferwithhydrogenperoxide.
Storeat2-8°Cuntilexpirationdate.
STP-100StoppingSolutionOnebottle,12mL,containing0.
2Msulfuricacid.
Storeat2to30°Cuntilexpirationdate.
WSH-100WashConcentrateAOnebottle,60mL,containingbufferedsalinewithanonionicdetergent.
Storeat2-30Cuntilexpirationdate.
Dilute25-foldwithdeionizedwaterpriortouse.
MATERIALSREQUIREDBUTNOTPROVIDED1.
Microtitrationplatereadercapableofabsorbancemeasurementat450nm,405nmand630nm.
2.
Microplateshaker.
3.
Microplatewasher.
4.
Semi-automated/manualprecisionpipettetodeliver10–250μL.
5.
Repeaterpipette.
6.
Vortexmixer.
7.
Deionizedwater.
WARNINGSANDPRECAUTIONSForin-vitroresearchuse.
Thefollowingprecautionsshouldbeobserved:a)Followgoodlaboratorypractice.
b)Usepersonalprotectiveequipment.
Wearlabcoatsanddisposablegloveswhenhandlingimmunoassaymaterials.
Page2of4DocumentNo:IFU.
AL.
113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISAc)HandleanddisposeofallreagentsandmaterialincompliancewithapplicableregulationsWARNING:PotentialBiohazardousMaterialThisreagentmaycontainsomeanimaland/orhumansourcematerial(e.
g.
serum)ormaterialsusedinconjunctionwithhumansourcematerials.
HandleallreagentsandpatientsamplesataBiosafetyLevel2,asrecommendedforanypotentiallyinfectioushumanmaterialintheCentersforDiseaseControl/NationalInstitutesofHealthmanual"BiosafetyinMicrobiologicalandBiomedicalLaboratories,"5thEdition,20071.
WARNING:PotentialChemicalHazardSomereagentsinthiskitcontainPro-Clean400andSodiumazide2asapreservative.
Pro-Clean400andSodiumAzideinconcentratedamountsareirritantstoskinandmucousmembranes.
Forfurtherinformationregardinghazardoussubstancesinthekit,pleaserefertotheMSDS,eitheratAnshLabs.
comorbyrequest.
SAMPLECOLLECTIONa)Serumistherecommendedsampletype.
b)Samplehandling,processing,andstoragerequirementsdependonthebrandofbloodcollectiontubethatyouuse.
Pleasereferencethemanufacturer'sinstructionsforguidance.
Eachlaboratoryshoulddeterminetheacceptabilityofitsownbloodcollectiontubesandserumseparationproducts.
c)Samplesmaybestoredat4°Cifassayedwithin24hours;otherwisesamplesmustbestoredat-20°Cor-80°Ctoavoidlossofbioactivityandcontamination.
d)Avoidassayinglipemic,hemolyzedorictericsamples.
e)Avoidrepeatedfreezingandthawingofsamples.
Thawsamplesnomorethan3times.
f)Forshipping,placespecimensinleakproofcontainersinbiohazardspecimenbagswithappropriatespecimenidentificationandtestrequisitioninformationintheoutsidepocketofthebiohazardspecimenbag.
FollowDOTandIATArequirementswhenshippingspecimens.
PROCEDURALNOTES1.
AthoroughunderstandingofthispackageinsertisnecessaryforsuccessfuluseoftheRatandMouseAMHELISAassay.
Itisthelaboratory'sresponsibilitytovalidatetheassayfortheiruse.
Accurateresultswillonlybeobtainedbyusingpreciselaboratorytechniquesandfollowingthepackageinsert.
2.
Acalibrationcurvemustbeincludedwitheachassay.
3.
Bringallkitreagentstoroomtemperaturebeforeuse.
Thoroughlymixthereagentsbeforeusebygentleinversion.
Donotmixvariouslotsofanykitcomponentanddonotuseanycomponentbeyondtheexpirationdate.
4.
Useacleandisposablepipettetipforeachreagent,calibrator,controlorsample.
Avoidmicrobialcontaminationofreagents,contaminationofthesubstratesolutionswiththeHRPconjugates.
Theenzymeusedasthelabelisinactivatedbyoxygen,andishighlysensitivetomicrobialcontamination,sodiumazide,hypochlorousacidandaromaticchlorohydrocarbonsoftenfoundinlaboratorywatersupplies.
Usedeionizedwater.
5.
Incompletewashingwilladverselyaffecttheoutcomeandassayprecision.
CareshouldbetakentoaddTMBintothewellstominimizepotentialassaydriftduetovariationintheTMBincubationtime.
Avoidexposureofthereagentstoexcessiveheatordirectsunlight.
PREPARATIONOFREAGENTS1.
AMHCalibratorsH:a.
TapandreconstituteRatandMouseAMHCalibratorHwith1mLdeionizedwater.
Solubilizefortenminutes,mixwellbeforeuse.
b.
PreparesevenpolystyrenetubesandlabelthemasCalA,CalB,CalC,CalD,CalE,CalFandCalG.
c.
Add500lofAMH/MISCalibratorA/SampleDiluenttoeachpolystyrenetubelabeledCalA-G.
d.
Add500lofreconstitutedAMHCalibratorH(fromstepa)tothetubelabeledCalG.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
e.
Add500lofCalG(fromstepd)tothetubelabeledCalF.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
f.
Add500lofCalF(fromstepe)tothetubelabeledCalE.
Vortexandmixthecontentinthetubethoroughlybeforethenextdilutiontransfer.
g.
Add500lofCalE(fromstepf)tothetubelabeledCalD.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
h.
Add500lofCalD(fromstepg)tothetubelabeledCalC.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
i.
Add500lofCalC(fromsteph)tothetubelabeledCalB.
Vortexandmixthecontentinthetubethoroughlybeforeuse.
j.
ThetubelabeledCalAcontains500lAMHCalibratorA/SampleDiluentandhas0AMHconcentrationsandshouldbeusedasBlank.
NOTE:Ifgreatersensitivityisdesired,CalBcanbefurtherdilutedtoB/2usingCalA.
k.
TheCalibratorsA-Hforinstanceshouldreadas0.
0ng/mL,0.
313ng/mL,0.
625ng/mL,1.
25ng/mL,2.
5ng/mL,5ng/mL,10ng/mLand20ng/mL.
AliquotandfreezetheRatandMouseAMHCalHStockimmediatelyformultipleuses.
Avoidrepeatedfreezethaws.
Frozenaliquotsat-20°Caregoodforoneyear.
l.
TheAMHconcentrationintheRatandMouseAMHcalibratorsHistraceabletothemanufacturer'sworkingcalibrators.
Valuesassignedbyothermethodologiesmaybedifferent.
Suchdifferences,ifpresent,maybecausedbyinter-methodbias.
2.
WashSolution:Dilutewashconcentrate25-foldwithdeionizedwater.
Thewashsolutionisstableforonemonthatroomtemperaturewhenstoredinatightlysealedbottle.
3.
MicrotitrationWells:Selectthenumberofcoatedwellsrequiredfortheassay.
Theremainingunusedwellsshouldbeplacedintheresealablepouchwithadesiccant.
Thepouchmustberesealedtoprotectfrommoisture.
SAMPLEPREPARATION1.
Maleratandmousesamples:Dilute1:50intoCalibratorA/SampleDiluent.
(1partunknownsampleinto49partsCalibratorA/SampleDiluent).
2.
Femaleratandmousesamples:Dilute1:10intoCalibratorA/SampleDiluent.
(1partunknownsampleinto9partsCalibratorA/SampleDiluent).
Page3of4DocumentNo:IFU.
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113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISANOTE:AMHvaluescanvaryfromonebreedtoanother.
Youmayneedtoestablishyourdilutionfactoraccordinglytoensurethedilutedsamplesfallwithinthemeasurablerangeoftheassay.
ASSAYPROCEDUREAllowallspecimensandreagentstoreachroomtemperature(23±2C)andmixthoroughlybygentleinversionbeforeuse.
NOTE:Allsamplesshouldbedilutedasperthe"SamplePreparation"section.
NOTE:Calibratorsanddilutedunknownsshouldbeassayedinduplicate.
NOTE:Alldilutedsamplesreadinghigherthanthehighestcalibratorshouldbefurtherdilutedinthe0ng/mLCalibratorA/Samplediluentpriortoassay.
1.
Labelthemicrotitrationstripstobeused.
2.
Pipette50μLoftheCalibrators(CalA-H)anddilutedunknowns(See"SamplePreparation"section)totheappropriatewells.
3.
Add50μLoftheAMHAssayBuffertoeachwellusingarepeaterpipette.
4.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for120minutesatroomtemperature(23±2C).
5.
Aspirateandwasheachstrip5timeswithWashSolution(350uL/well)usinganautomaticmicroplatewasher.
6.
Add100μLoftheAntibody-BiotinConjugateRTUtoeachwellusingarepeaterpipette.
7.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for60minutesatroomtemperature(23±2C).
8.
Aspirateandwasheachstrip5timeswiththeWashSolution(350uL/well)usinganautomaticmicroplatewasher.
9.
Add100LoftheStreptavidin-EnzymeConjugate-RTUtoeachwellusingarepeaterpipette.
10.
Incubatetheplate,shakingatafastspeed(600-800rpm)onanorbitalmicroplateshaker,for30minutesatroomtemperature(23±2C).
11.
Aspirateandwasheachstrip5timeswiththeWashSolution(350uL/well)usinganautomaticmicroplatewasher.
12.
Add100μLoftheTMBchromogensolutiontoeachwellusingarepeaterpipette.
Avoidexposuretodirectsunlight.
13.
Incubatethewells,shakingat600–800rpmonanorbitalmicroplateshaker,for8-12minatroomtemperature(23±2°C).
NOTE:Visuallymonitorthecolordevelopmenttooptimizetheincubationtime.
14.
Add100μLoftheStoppingsolutiontoeachwellusingarepeaterpipette.
Readtheabsorbanceofthesolutioninthewellswithin20minutes,usingamicroplatereadersetto450nm.
NOTE:Zerocalibratorshouldbeprogrammedas"Blank"whilereadingtheopticaldensity.
Ifinstrumenthasawavelengthcorrection,settheinstrumenttodualwavelengthmeasurementat450nmwithbackgroundwavelengthcorrectionat630nm.
RESULTSNOTE:Theresultsinthispackageinsertwerecalculatedbyplottingthelogopticaldensity(OD)dataonthey-axisandlogAMHconcentrationonX-axisusingacubicregressioncurve-fit.
Alternatively,logvs.
logquadraticregressioncurve-fitcanbeused.
Otherdatareductionmethodsmaygiveslightlydifferentresults.
1.
Optimumresultscanbeobtainedatincubationtemperatureof23±2°C.
2.
Calculatethemeanopticaldensity(OD)foreachcalibrator,Control,orUnknown.
3.
PlotthelogofthemeanODreadingsforeachoftheCalibratorsalongthey-axisversuslogoftheAMHconcentrationsinng/mLalongthex-axis,usingacubicregressioncurve-fit.
4.
DeterminetheAMHconcentrationsoftheunknownsfromthecalibrationcurvebymatchingtheirmeanODreadingswiththecorrespondingAMHconcentrations.
5.
AnysamplereadinghigherthanthehighestCalibratorshouldbeappropriatelydilutedwiththe0ng/mL(CALA/SampleDiluent)andre-assayed.
6.
Anysamplereadinglowerthantheanalyticalsensitivityshouldbereportedassuch.
7.
Multiplytherecoveredunknownvaluesbytheappropriatedilutionfactor.
LIMITATIONSThereagentssuppliedinthiskitareoptimizedtomeasureAMHlevelsinratandmouseserum.
Ifthereisevidenceofmicrobialcontaminationorexcessiveturbidityinareagent,discardthevial.
Forassaysemployingantibodies,thepossibilityexistsforinterferencebyheterophileantibodiesinthesamples4.
QUALITYCONTROLEachlaboratoryshouldestablishmeanvaluesandacceptablerangestoassureproperperformance.
EachlaboratoryshouldestablishinternalAMHcontrolsranges.
Theresultsshouldfallwithinestablishedconfidencelimits.
Afullcalibrationcurve,andcontrol,shouldbeincludedineachassay.
TMBshouldbecolorless.
Developmentofanycolormayindicatereagentcontaminationorinstability.
REPRESENTATIVECALIBRATIONCURVEDATAWellNumberWellContentsMeanAbsorbanceConc(ng/mL)A1,A2CalibratorsA0.
060(Blank)0B1,B2B0.
060.
336C1,C2C0.
1360.
672D1,D2D0.
2521.
344E1,E2E0.
5112.
687F1,F2F0.
9885.
375G1,G2G1.
93410.
75H1,H2H3.
41921.
5CAUTION:Theabovedatamustnotbeemployedinlieuofdataobtainedbytheuserinthelaboratory.
NOTE:TheODvalueslistedaboveforcalibratorsB-Hareblanksubtracted.
ANALYTICALCHARACTERISTICSAllanalyticalcharacteristicsarestatedinng/mL(1ng/mLAMH=7.
14pmol/L)Specificity:TheAnshLabsRatandMouseAMHassayhasbeenscreenedforcrossreactivitywithotherspeciesAMH,including:rabbit,avian,hamster,belugawhale,andbottlenosedolphin.
TheantibodiesexhibithighaffinitytoAMHofthesespecies.
TestsforparallelismbylinearlydilutingspecimensfromthesespeciesandassayingthedilutedserumdemonstratetheELISAkitaccuracyfordeterminingAMHintheseanimalsLinearity:MultipledilutionsofsamplescontainingvariousAMH/MISlevelsweredilutedwithCalibratorA/samplediluent.
The%recoveryonindividualsamplesisrepresentedinthefollowingtable.
SampleDilutionFactorExpectedConc.
(ng/mL)ObservedConc.
(ng/mL)%RecoveryHamster1:101:201:401:802.
21.
10.
550.
27NA1.
10.
680.
31NA100%124%115%Mouse11:201:401:8020.
66510.
3335.
166NA9.
8654.
771NA95%92%Page4of4DocumentNo:IFU.
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113RevisionNo:05ReleaseDate:10/10/2016RatandMouseAMHELISA1:1602.
5832.
40393%Mouse21:101:201:4021.
69610.
8485.
424NA11.
1715.
710NA103%105%Mouse31:101:201:4019.
7399.
8704.
935NA10.
3285.
206NA105%105%RecombinantRatAMHNeat1:021:041:089.
934.
972.
481.
24NA4.
782.
211.
04NA96%89%84%REFERENCES1.
HHSPublication,5thed.
,2007.
BiosafetyinMicrobiologicalandBiomedicalLaboratories.
Availablehttp://www.
cdc.
gov/biosafety/publications/bmbl5/BMBL52.
DHHS(NIOSH)PublicationNo.
78–127,August1976.
CurrentIntelligenceBulletin13-ExplosiveAzideHazard.
Availablehttp://www.
cdc.
gov/niosh.
3.
ApprovedGuideline–ProceduresfortheHandlingandProcessingofBloodSpecimens,H18-A3.
2004.
ClinicalandLaboratoryStandardsInstitute.
4.
KrickaL.
Interferencesinimmunoassays–stillathreat.
ClinChem2000;46:1037–1038.
TheAnshLabslogoisatrademarkofAnshLabs.
Manufacturedby:AnshLabs445MedicalCenterBlvd.
Webster,TX77598-4217,U.
S.
A.
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