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Mission StatementDiapharma Group, Inc. in West Chester, Ohio sells hemostasis, thrombosis, platelet function testing, instrumentation, and apoptosis products in the diagnostic and research fields and provides strong technical competence and experienc...
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Diapharma/Chromogenix S-2765™/S821413/Vial/25mg

Diapharma/Chromogenix S-2765™/S821413/Vial/25mg
  • Diapharma/Chromogenix S-2765™/S821413/Vial/25mg
商品介绍

Description:

ChromogenixS-2765™isachromogenicsubstratefordeterminationofFactorXaactivity.Itisalsoverysensitivetotrypsin.

S-2765™ issuitable formeasuringFXainhibition inheparinanti-Xaassaysandantithrombinanti-Xaassays.

Composition:

EachvialcontainsthechromogenicsubstrateS-2765™,25mgandmannitol60mgasabulkingagent.

StABIlity:Lyophilizedsubstance:stableat25°Cuntilexpirydateprintedonthelabel.Thesubstanceishygroscopicandshouldbestoredinadryplace.

Solution:2mmol/LinH2Oisstableforsixmonthsat2to8°C.Suitablestocksolution:1-2mmol/LinH2O.

Chemicalname:N-a-Benzyloxycarbonyl-Darginyl-L-glycyl-L-arginine-pnitroaniline-dihydrochloride

Formula:N-a-Z-D-Arg-Gly-Arg-pNA·2HCl

Mol.wt.:714.6

chromogenix chromogenic substrate assay test kit

FactorXaIUandEnzymeActivity:

FactorXa,whichhasamolecularweightof44KDa,istheactivatedformofFactorX(MW:59KDa).

TheInternationalUnitsofFactorXcorrespondtotheamountofFactorXcontainedin1mlofnormalplasma.Thisisabout8mg/lor0.13µmol/l.

SincethereisnoWHOstandardforFXa,onewouldassumethatifalltheFactorXinnormalplasmawasconvertedtotheactivatedform,theFactorXaconcentrationwouldbeapproximately5.7mg/l.

TheactivityofhumanFactorXaascalculatedfromthekinetictablesis1.5nkat/µgwiththechromogenicsubstrateS-2222™,and4.4µkat/µgwiththechromogenicsubstrateS-2765™.

Theactivityof1µgofFactorXaasdeterminedbyFrieberger(1)is1.9nkatchromogenicsubstrateS-2222™.

Thus,1plasmaequivalentunitofFactorXwouldcorrespondto15.2nkatchromogenicsubstrateS-2222™.

FribergerPetal.
Syntheticpeptidesubstrateassaysandfibrinolysisandtheirapplicationonautomates.In:SeminarsinThrombosisandHaemostasis,Vol.9,281-300(1983).

FactorXMethod:

DeterminationoffactorXinplasmawithChromogenicSubstrateS-2765™

MeasurementPrinciple

Themethodisbasedonatwo-stageprinciple.Instageone,FactorXisactivatedinthepresenceofcalciumtoFactorXa(FXa)usingtheactivatorRussell’sVipervenom(RVV).Instagetwo,thegeneratedFXahydrolysesthechromogenicsubstrateZ-D-Arg-Gly-Arg-pNA(S-2765™),thusliberatingthechromophoricgrouppNA(p-nitroaniline).Thecoloristhenreadphotometricallyat405nm.ThegeneratedFXa(andthustheintensityofcolor)isproportionaltotheFXactivityofthesample.

FactorXRVV
FactorXa
Z-D-Arg-Gly-Arg-pNA+H2OFXa
Z-D-Arg-Gly-Arg-OH+pNA

Reagents

  1. S-2765™,25mgArt.No.S821413
    ReconstitutethesubstrateS-2765™(MW:714.6)with20mlsterilewater.
  2. Russell’sViperVenom(RVV)
    PrepareasolutionofRussell’sViperVenomataconcentrationof0.087mg/ml.
  3. CaCl2
    0.1mol/lcalciumchloridesolution.
  4. TrisEDTABuffer
    Dilutethebuffer1:10withdistilledwateraccordingtotheinsertsheetinstructions.
  5. NormalPlasma
    Calibrated,lyophilizedorfreshfrozenhumanplasmaisusedforthestandardizationoftheassay.Apoolednormalplasmacanbepreparedbytakingsamplesfrom20healthydonors.10-30mlcitrateblood(9volbloodand1vol0.1mol/lsodiumcitrate)fromeachdonoriscentrifugedat2000xgfor20minutesat15-25°C.Theplasmaispooledandsubsequentlydispensedinsmallvolumes,whicharefrozenrapidlyat-20°Corbelow.Toavoidlowtemperatureactivationofprekallikreintheplasmaiskeptat15-25°Cbeforeuseorfreezing.Thawingofplasmashouldbeperformedat37°Candthenkeptat15-25°Cuntilused.
  6. RVV+CaCl2
    Beforeuse,mix1volumeofRVVwith1volumeofCaCl2.Themixtureisstablefor48hoursat2-8°C.
  7. Aceticacid20%
    Aceticacidisusedasastoppersolutionintheend-pointmethod.

Specimencollection

Blood(9vol)ismixedwith0.1mol/lsodiumcitrate(1vol)andcentrifugedat2000xgfor20minutesat20-25°C.Storage:1weekat2-8°Cor3-4monthsat-20°C.


Standardcurve

Predilution

FinalDilution

FX%NormalPlasma mlBuffer mlPredilplasma mlBuffer ml
01000
252575501000
505050501000
757525501000
100501000
12450800

Method

SampleDilution
Buffer1000
Testplasmaorstandard50
Mix

AcidStoppedMethodAB
DilutedSample200 ml50 ml
Incubateat37°C3-4min3-4min
Substrate(37°C)200 ml50 ml
Mixandaddwithin30sec
RVV+ CaL2200 ml50 ml
Mixandincubateat37°C3min3min
Aceticacid20%200 ml50 ml

A=testtubemethod
B=microplatemethod

Sampleblankactivitiesshouldbedeterminedandsubtractedwhenanalyzingstronglycoloredplasma,e.g.lipemicandhemolytic.Thesampleblanksarepreparedbymixingthedilutedsample,aceticacid20%andwaterinsteadofthereagents(400µlfortesttubesand100µlformicroplates).Readtheabsorbanceofthesamplesandblanksat405nm.Thecolorisstableforatleastfourhours.WhenpossIBLe,useadualwavelengthmodewith490nmasthereferencewavelength.


Initialratemethod

Whenperformingtheinitialratemethod,transferthemicroplatetoamicroplatereaderimmediatelyaftertheadditionofRVV+CaCl2andreadthechangeinA/min.Themicroplatereadermustbepre-incubatedat37°C.


Calculation

PlotAorΔA/minforthestandardsagainsttheirconcentrationofFactorX.ReadtheFactorXvalueforthecorrespondingAorΔA/minoftheunknowntestsamplefromthestandardcurve.


Bibliography

  1. KieselWetal.FactorXactivatingenzymefromRussell’sViperVenom;isolationandcharacterisation.Biochemistry15,4901-4905(1976).
  2. LindhoutMJetal.ActivationofdecarboxyfactorXbyaproteinfromRussell’sViperVenom.PurificationandpartialcharacterisationofactivateddecarboxyfactorX.BiochemBiophysActa533,327-341,(1978).
  3. BergströmKandEgbergN.DeterminationofvitaminKsensitivecoagulationfactorsinplasma.Studiesonthreemethodsusingsyntheticchromogenicsubstrates.ThrombRes12,531-547(1978).
  4. VanWijkEMetal.ArapidmanualchromogenicfactorXassay.ThrombRes22,681-686(1981).EgbergNandHeedmanPA.SimplifiedperformanceofamidoliticfactorXassay.ThrombRes25,437-440(1982).
  5. ChabbatJetal.AprotininisacompetitiveinhibitorofthefactorVIIa-tissuefactorcomplex.ThrombRes71,205-215(1993).
  6. MielickiWPandGordonSG.ThreestagechromogenicassayfortheanalysisofactivationpropertiesoffactorXbycancerprocoagulant.BloodCoagulFibrinol4,441-446(1993).
  7. KoppakaVetal.SolublephospholipidsenhancefactorXa-catalyzedprothrombinactivationinsolution.Biochemistry35,7482-7491(1996).
  8. RiesbeckKetal.HumantissuefactorpathwayinhibitorfusedtoCD4bindsbothFXaandTF/FVIIaatthecellsurface.ThrombHaemost78,1488-1494(1997).
  9. RomischJetal.Comparativeinvitroinvestigationofprothrombincomplexconcentrates.SeminThrombHemost24,175-181(1998)
  10. FariaF,etal.AnewfactorXainhibitor(lefaxin)fromtheHaementeriadepressaleec.ThrombHaemost82,1469-73(1999).
品牌介绍

Mission Statement

Diapharma Group, Inc. in West Chester, Ohio sells hemostasis, thrombosis, platelet function testing, instrumentation, and apoptosis products in the diagnostic and research fields and provides strong technical competence and experience to ensure customer expectations will be met or exceeded.

diapharma hemostasis chromogenic clotting elisa assay test kit

 

History

The Diapharma Group, Inc. formed on January 1, 1997 from Pharmacia Hepar, Inc. in Franklin, Ohio, and began as the exclusive US and Canadian distributor of the Chromogenix substrates and assays.

Over a quarter-century ago, Chromogenix developed the first chromogenic substrate technology under the former name, Kabi Diagnostica. Kabi later merged with Pharmacia. Some of Diapharma’s current staff worked inside the Chromogenix department within Pharmacia’s heparin manufacturing plant.

In 1998, Diapharma moved to West Chester, Ohio, where it remains today. Over the years, Diapharma expanded its product line to include a variety of hemostasis, cell death, platelet function, ecotoxicology, assays, reagents, antibodies and instruments from superior manufacturers.

In 2017, Diapharma celebrated twenty years of success


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