ThiskitmeasuresPRAandtheresultsareexpressedintermsofmassofangiotensin-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.

CLINICALAPPLICATIONS
MeasurementofPRAisimportantfortheclinicalevaluationofhypertensivepatients.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.