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IBL/Angiotensin I (Plasma Renin activity) ELISA/DB52011/

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Kitsize192(96)MethodELISAIncubationtime(90min)+1h15minStandardrange0.2-60ng/mLSpecimen/Volumes500µLEDTAPasmaSubstrate/isotopeTMB450nmRegulatoryStatus:EU:CEDetailsfor: AngiotensinI(PlasmaReninactivity)ELISAThiskitmeasuresPRAandtheresultsareexpressedintermsofmassofangiotensin-I(Ang-I)generatedpervolumeofhumanplasmainunittime(ng/mL.h).ThebloodsampleiscollectedinatubethatcontainsEDTA.Theplasmaisseparatedandeitherstoredfrozenorkeptatroomtemperatureforimmediateuse,samplesshouldnotbechilledoniceorstoredattemperaturesbetween0and10°CduringcollectionorprocessingbeforeadjustmentofpH,thiscouldleadtooverestimationofreninactivity.BeforethestartofimmunoassayaproteaseinhibitorandtheGenerationbufferisaddedtotheplasmasample,whichwillpreventAngiotensin-I(Ang-I)inplasmafromdegradation.ThepHoftheplasmasampleshouldbearound6.0aftertheadditionofthesuppliedGenerationbuffer.Theplasmasampleissplitintwoandthefractionsareincubatedat0-4°C(inicebath)and37oCrespectivelyfor90minutesorlonger,toallowthegenerationofAng-Ibyplasmareninat37°C.Optionally,thepHcanbeadjustedto6.5or7.4.AdjustmentofpHisacriticalstepduringtheassay,acidificationofplasmatopH3.3orlowerforprolongedtimewithsubsequentreturntoneutralpHcausesirreversIBLeactivationoftherenin(Derkxetal.,1987),ontheothersideincubationatpHhigherthan8.0candestroyrenin.Duringtheimmunoassayincubation,anothersetofproteaseinhibitorsareinvolved,whichfunctiontostopthenewgenerationaswellasdegradationofAng-Itosmallerpeptides.TheimmunoassayofAng-Iisacompetitiveassaythatusestwoincubations,withatotalassayincubationtimeoflessthantwohours.DuringthefirstincubationunlabelledAng-I(presentinthestandards,controlsandplasmasamples)competeswithbiotinylatedAng-Itobindtotheanti-Ang-Iantibody.InthesecondincubationthelabelledStreptavidin-HRPconjugate,bindstotheimmobilizedAng-I-Biotin.Thewashinganddecantingproceduresremoveunboundmaterials.ThecolorimetricHRPsubstrateisaddedand,afterstoppingthecolordevelopmentreaction,thelightabsorbance(OD)ismeasuredinamicrowellplatereader.TheabsorbancevaluesareinverselyproportionaltotheconcentrationofAng-Iinthesample.AsetofcalibratorsisusedtoplotastandardcurvefromwhichtheconcentrationsofAng-Iinthesamplesandcontrolscanbedirectlyread.CLINICALAPPLICATIONSMeasurementofPRAisimportantfortheclinicalevaluationofhypertensivepatients.Inparticular,determinationofplasmareninactivitycanhelpinthediagnosisofprimaryhyperaldosteronism(5-13%ofhypertensivecases)andassistinthetherapyandmanagementofotherformsofhypertension.PRA,incontrasttothedeterminationofreninconcentration,isamoreaccurateindicatorofprimaryhyperaldosteronism(PHA),becauseofseveralreasons:1.PRAistheexpressionoftherateofAng-Iformationthroughtheenzymaticactionofreninonitssubstrate,angiotensinogen,thereforePRAdependsnotonlyonreninconcentrationbutalsoontheconcentrationofangiotensinogenwhichisignoredinthereninconcentrationassay;2.Plasmareninconcentrationassaydoesnotensuresensitivityinlowreninstates,whilethesensitivityofthePRAassaycanbeenhancedbyincreasingtheincubationtimeduringthegenerationstep(Sealeyetal.,2005),3.WhenaninhibitorisboundtothereninactivesitePRAisinhibited,whereasthepresenceoftheinhibitordoesnotaffecttherecognitionofreninbycurrentlyavailableimmunoassays,thereforetotalreninconcentrationdoesnotalwayscorrelatewithplasmareninactivity(Campbelletal.,2009).Reninliberatesangiotensin-Ifromangiotensinogen.Angiotensin-Iistransformedtoangiotensin-IIlargelyinpulmonarycirculationbyangiotensinconvertingenzyme(ACE).Angiotensin-IIraisesbloodpressurebydirectarteriolarvasoconstriction,promotingsodiumretension,andstimulatingthesecretionofaldosteronefromtheadrenalcortex.Aldosteronealsoexertsaneffecttorestoresodiumbalanceandliftarterialpressure.Accuratemeasurementoftheconcentrationofcirculatingangiotensin-IIischallengingbecauseofitsinstABIlityinbloodsamples.AldosteroneconcentrationcanbeeasilydeterminedusingtheIBLimmunoassaykit(RE52301).ThisproductrepresentstherightalternativetothesoondiscontinuedRenin(PRA)RIAfromDiaSorin(REN-CTK).Pleasecontactusifyoushouldneedanykitfortrialpurposes!Orifyoushouldneedanysupportregardingcomparisonstudies.Wewillbehappytoprovideyouwithexpectedhelp.ForconcretedatapleaseconsulttheInstructionforUseinthedownloadboxontherightside.

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