Description:

Technozym®ADAMTS-13ActivityisachromogenicELISAkitforthedeterminationofADAMTS-13activityinhumanplasma.ADAMTS-13isanenzymethatspecificallycleavesunusuallylargevonWillebrandFactormultimers,whichinduceplateletthrombusformationunderhighshearstress.IftheactivityofADAMTS-13isloweredforsomereason,unusuallylargevWFmultimersmayaccumulate.

Advantages:

• Easytousein-houseassaycanleadtosignificantcostsavings
• Noneedforanyspecialtechniqueorinstrument,juststandardELISAequipment
• ADAMTS-13activitysensitivitydownto0.5%ofnormals
• Shortassaytime(3-4h)
• Calibratorsandcontrolsincluded
• Nointerferencesfrombilirubin,hemoglobinorelastase

KitComposition:

Reagents, Composition,StorageandStABIlity

• ELISAteststrips(12),with8wellseach,coatedwithamonoclonalantiGST-Antibody.Thedryingagentissuppliedinanaluminiumbag.Stabilityafterreconstitution:expirydate.
• GST-vWF73Substrate;2vials;lyophilized;6mL.Stabilityafterreconstitution:6weeks.
• Calibrators(standards)numberedfrom1to6;lyophilized;1vialeach;0.5mL.Stabilityafterreconstitution:6months.Concentrationsarelot-specific;consultthelabelonthevial.
• Highandlowcontrolplasma,lyophilized,1vialeach,0.5mL.Stabilityafterreconstitution:6months.Concentrationsarelot-specific;consultthelabelonthevial.
• Reactionbuffer;1vial;30mL;readytouse.Stabilityafterreconstitution:6months.
• Conjugate:HRPconjugatedmonoclonalanti-N10Antibody:1vial;12mL;readytouse.Stabilityafterreconstitution:1month.
• ColorreagentTMB(Tetramethylbenzidine);1vial;12mL;readytouse.Stabilityafterreconstitution:expirydate.
• WashBufferconcentrate,10-foldconcentrated,1vial,32mL.Stabilityafterreconstitution:6months.
• Stopsolution;sulphuricacid0.5mol/L,1vial,12mL;readytouse.Stabilityafterreconstitution:expirydate.
• SampledilutionMicroplate,1plate(ONLYforsampledilution!).

Theexpirydateprintedonthelabelsappliestostorageoftheunopenedvialat2-8°C.

AssayPrinciple:

Background:

ADAMTS-13

ADAMTS-13(adisintegrin-likeandmetalloproteinasewiththrombospondintype1motif13)isanenzyme(vWF-cleavingproteaseorvWF-CP)thatspecificallycleavesvonWillebrandFactor(vWF)multimers.

ManyoftheoriginalassaysformeasuringADAMTS-13activityinvolvedusingamultimericvWFsubstratethatwaseitherplasma-derivedorrecombinant.Theseassaysaretimeconsuming(from2to4days)anddifficulttoreproducebetweentestingcenters.ManyoftheissueswerecircumventedbythedevelopmentvWFpeptidesubstrates,leADIngtoaturn-aroundtimefrom1to4hours.ADAMTS-13substrates(ie,FRETsubstrates)wereeventuallydevelopedintocommercialkits.However,differencesinthetechnologyusedformeasuringADAMTS-13activityinthekitsmayresultinlimitationsinvariousproductsgivingdiscrepantresults.SomelimitationsinanymethodformeasuringADAMTS-13activitymaybeunavoidable;however,lackofinterferencesandaccuratemeasurementatverylowADAMTS-13activityarecriticalinlaboratoryassays.

Fluorescentassaysalsohavenumerouslimitations,suchasinterferencesfromplasmaproteinssuchashemoglobinandbilirubin.Forexample,bilirubinabsorbslightatthesamewavelengthsasthechromophoresinFRETS-vWF73,andhemoglobinabsorbsat550nmandalsodirectlyinhibitsADAMTS-13regardlessoftheassaymethod.ThereissomerecentevidencethatunconjugatedbilirubincandirectlyinhibitADAMTS-13aswell.It’spossIBLetolessentheseissuesbydilution,butthismayresultinlimitingassaysensitivity,whichiscriticalforADAMTS-13assays.

LiquidphaseFRETassaysarealsosubjecttodiscrepanciesbecauseofcleavageoftheADAMTS-13substrate(ie,vWF73)byotherproteases.Forexample,thepresenceofelastaseinasamplemixedwiththesubstratewillresultincleavageleadingtoameasuredfluorescentsignal,whenanypeptidylbondbetweenthe2fluorescentdyesiscleaved.WhilelevelsofADAMTS-13intheplasmasamplewillalwaysvarydependingonthediseasecondition(whichmayresultinabnormallevelsofmanydifferentproteasesandotherproteins),proteaseinterferencecanbereducedduringtheactualassayiftheADAMTS-13isfirstcapturedonaplatefollowedbywashingbeforeaddingtheFRETsubstrate.ELISAplate-basedmethodscanreduceoreliminatetheseinterferences,asthesampleiswashedaway.

AnadditionalalternativeistoavoidfluorescentassaysusingastandardchromogenicELISAmethod(HRP/TMBreagents)thatrequiresstandardELISAequipmentratherthanafluorescentreader.PeptidevWFsubstratesarestillusedbutauniquemethodthatcapturesthesubstrateontheplateand,withADAMTS-13fromthesample,cleavesthepeptidetoexposeaneo-epitope.ThisnewepitopecanberecognizedbyaspecificantibodyallowingADAMTS-13activitytobedeterminedbyELISA. Ahighsampledilutioncanavoidbilirubinandhemoglobininterference.Inaddition,proteaseinterferenceisminimizedusinganantibodyveryspecifictotheADAMTS-13cleavagesite(Tyr1605-Met1606),andmostproteases(ie,elastase)cleaveadifferentsite(ie,M1606-V1607orV1607-T1608).OnlycathepsinGmayhavethesamecleavagesite.Usingthistypeofassay,sensitivitydownto0.5%ADAMTS-13activitycanbereached.