Detection and Quantification of Gram Negative Bacterial Endotoxin ...
Transcript of Detection and Quantification of Gram Negative Bacterial Endotoxin ...
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 1/14
DetectionandQuantificationofGramNegativeBacterialEndotoxinContaminationinNanoparticleFormulationsbyKineticTurbidimetricMicroplate
LALAssay
AUTHORED BY: DATE:
Rainer Ossig 14.04.2016
Matthias Rösslein 01.10.2016
REVIEWED BY: DATE:
Matthias Rösslein 13.07.2016
Matthias Rösslein 01.10.2016
APPROVED BY: DATE:
Matthias Rösslein 13.07.2016
Matthias Rösslein 01.10.2016
DOCUMENTHISTORY
Effective Date Date Revision Required Supersedes
01.10.2016 01.10.2016 13.07.2016
Version Approval Date Description of the Change Author / Changed by
1.0 13.07.2016 All Initial Document Rainer Ossig
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 2/14
Version Approval Date Description of the Change Author / Changed by
1.1 01.10.2016 Chapter 7 Updated Acceptance criteria now in line with NCI-NCL acceptance criteria
Matthias Rösslein
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 3/14
TableofContent1 Introduction......................................................................................................................................4
2 PrincipleoftheMethod....................................................................................................................4
3 ApplicabilityandLimitations(Scope)...............................................................................................4
4 RelatedDocuments..........................................................................................................................4
5 EquipmentandReagents..................................................................................................................5
5.1 Equipment.................................................................................................................................5
5.2 Reagents....................................................................................................................................5
5.3 ReagentPreparation..................................................................................................................6
5.3.1 PreparationofPyrotell-TLALReagent...............................................................................6
5.3.2 EndotoxinControlStandardEndotoxinstocksolution.......................................................6
5.3.3 PreparationofEndotoxincalibrationstandards.................................................................7
5.4 AssayControlReactions............................................................................................................7
5.4.1 PreparationofInhibition/EnhancementtestusingaPositiveProductControl(PPC)........7
5.4.2 QualityControls..................................................................................................................8
5.4.3 NegativeControl.................................................................................................................9
6 Procedure.........................................................................................................................................9
6.1 Generalremarks........................................................................................................................9
6.2 PreparationofStudySamples...................................................................................................9
6.3 Flowchart................................................................................................................................10
6.4 MeasurementProcedure........................................................................................................11
6.4.1 Testprocedure.................................................................................................................11
6.4.2 Preparationofthe96-wellmicroplate.............................................................................11
6.5 DataAnalysis...........................................................................................................................12
7 AssayAcceptanceCriteria..............................................................................................................13
8 HealthandSafetyWarnings,CautionsandWasteTreatment.......................................................13
9 Abbreviations.................................................................................................................................13
10 References....................................................................................................................................14
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 4/14
1 IntroductionThisdocumentdescribesaprotocolforaquantitativedetectionofGramnegativebacterialendotoxininnanoparticlepreparationsusingakineticturbidimetricLimulusAmebocyteLysate(LAL)assay.PrincipleoftheMethod
2 PrincipleoftheMethodThismethodreliesonaninvitroend-productendotoxintestwhichutilizesaLimulusAmebocyteLysate(LAL),anextractofbloodcells(amebocytes)fromthehorseshoecrab.Themethodisdesignedtodetectendotoxinactivityphotometricallywithanautomatedmicroplatereaderincubatingthereactionmixtureatcontrolledtemperatureof37°C.
GramnegativebacterialendotoxincatalyzestheactivationofproenzymeintheLimulusAmebocyteLysate.Bang1observedin1956thattheinfectionofthehorseshoecrabLimuluspolyphemuswithGram-negativebacteriaresultedinintravascularcoagulation,asaresultofareactionbetweenendotoxinandaclottingproteininamebocytesofLimulus2.ThemethodisbasedtheinitialreactionoftheLALwithendotoxin.ALALproenzymeisactivatedinthepresenceofendotoxin.Asaresultofthefollowingcascadeofenzymeactivationstepscoagulationisinitiatedandturbidityofthereactionmixtureincreases.Thedevelopmentofturbidityismeasuredusinganautomatedplatereaderandthetimetoreachaspecificincrementofturbidity(theonsettime)isdetermined.Higherendotoxinconcentrationsgiveshorteronsettimes.ConcentrationofendotoxininasampleiscalculatedfromastandardcurvepreparedbytheonsettimeofknownconcentrationofendotoxinstandardintoLALgradewater.ThismethodreliesonLimulusAmebocyteLysatePYROTELL®–TbyPyroquantDiagnostikGmbH,asubsidiaryofAssociatesofCapeCod,Inc.(ACC)3.DataanalysisisperformedusingMARSsoftware(BMGLabtechGmbH).
TheamountofendotoxinpresentwhichiscalculatedfromastandardcurvepreparedbydilutionofanendotoxinstandardofknownconcentrationsofintoLALgradewater.
3 ApplicabilityandLimitations(Scope)ThisSOPwasdevelopedtodetermineandquantifyendotoxincontaminationofdifferentnanomaterials.ThisSOPwascreatedaccordingtoDINENISO297014adaptedfortheanalysisnanomaterials4.AndaccordingtothesuppliersinstructionfortheuseoftheKineticTurbidimetricMicroplateLALAssayPyrotell-T3.UsePyrotell-Tforinvitrodiagnosticpurposesonly.Donotuseitforthedetectionofendotoxemia3.
4 RelatedDocumentsTable1:
DocumentID DocumentTitleNCLMethodSTE-1.2 DetectionandQuantificationofGramNegativeBacterialEndotoxin
ContaminationinNanoparticleFormulationsbyKineticTurbidityLALAssay
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 5/14
5 EquipmentandReagents
5.1 Equipment5.1.1 Pyrogen-freemicrocentrifugetubes,1.5mL(e.g.EppendorfBioPure®)
5.1.2 Pyrogen-freepipettesandbarriertipscoveringtherangefrom0.01to1mL(e.g.SarstedtBiosphere®)
5.1.3 Pyrogen-freedispensertips,100µlincrement(e.g.EppendorfBioPure®)
5.1.4 Repeatpipettororeight-chanelpipettor
5.1.5 96wellplate,pyrogen-free(e.g.Costar3596,ACCPYROPLATE®orequivalent)
5.1.6 Disposableendotoxin-freeglassdilutiontubes13×100mm(LonzaN207)or 12x75mm(ACCTB240)orequivalent
5.1.7 Reagentreservoirs(Lonza00190035orequivalent)
5.1.8 Microcentrifuge
5.1.9 Refrigerator,2-8°C
5.1.10 Freezer,-20°C
5.1.11 Vortexmixer
5.1.12 Parafilm®“M”Laboratoryfilm(PechineyPlasticPackaging)
5.1.13 automatedMicroplatereader,temperaturecontrolled37°C,at340nmabsorption(e.g.Novostar®,ClarioStar®,BMGLabtechGmbH)
5.2 Reagents5.2.1 Testnanomaterial
5.2.2 LIMULUSAMEBOCYTELYSATEPYROTELL®–TForTheDetectionAndQuantificationOfGram NegativeBacterialEndotoxins(PYROQUANTDIAGNOSTIK,AssociatesofCapeCod,Inc.(ACC))
5.2.3 ControlStandardEndotoxin(CSE)(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.4 Glucashield®(1→3)-ß-D-GlucanInhibitingBuffer(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.5 LALReagentWater(PYROQUANTDIAGNOSTIKGmbH,ACC)
5.2.6 Sodiumhydroxide,0,1N,endotoxinfree(e.g.Acila1712200)
5.2.7 Hydrochloricacid0,1N,endotoxinfree(e.g.Acila1712300)
5.2.8 Endotoxinfreewater(e.g.ACCW0051;Acila1715050;orequivalent)
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5.3 ReagentPreparationStoreallprovidedreagentsofthekitat2–8°C.Priortouseallowreagentstoequilibratetoroomtemperature3.
5.3.1 PreparationofPyrotell-TLALReagentTheassayreagentisprovidedaslyophilizedmixtureofLALlysateItisreconstitutedaccordingtomanufacturer’srecommendations,andcanbeperformedinLALgradewater(LALReagentWater)orGlucashield®Bufferavailablefromthesupplierasseparatecomponents.EUNCLpreferredmethodistheapplicationofGlucashield®bufferwhichallowstoexcludeinterferencefromβ-1,3-Glucanswhichisverycommoninnanomaterialsproducedusingfiltrationsteps.Substancescontainingβ-1,3-Glucansareimportantsourcesoffalse-positivesandasynergisticresponse(i.e.enhancement)isfrequentlyseenwithβ-Glucancontainingsamplesspikedwithendotoxin.TheusageofaGlucashield®bufferisthereforeindicatedwheneverβ-1,3-Glucancontaminationisexpected3.
ReconstitutePyrotell-TLALReagentonlyimmediatelybeforeuse.Addthevolumeasindicatedontheviallabel,whichusuallyis5mL,andisgoodtoperform48singlereactions.Incaseforalargernumberofsamplesmorethanonevialisrequired,poolreconstitutedreagentoftwoorseveralvialsbeforeuse.Exerciseextremecautiontoavoidformationofairbubbles.Donotvortexthereconstitutedlysate.Pipettewithcaution.Mixonlybyverygentlyswiveltoavoidfoaming.Incaseofbubblesallowtoclearbeforeuse.CoverthevialwithParafilmM®whennotinuse.
StorethelyophilizedPyrotell-TLALReagentat-20to8°Cuntilexpirationdateonthelabel.ReconstitutedLALReagentshouldbeusedpromptlyandisstableforupto24hoursat2to8°C.,orcanbestoredatorbelow-20°Corcolderforuptothreemonthsiffrozenimmediatelyafterreconstitution.FreezeandthawthereconstitutedLALReagentonlyonce(ACC)3.
5.3.2 EndotoxinControlStandardEndotoxinstocksolutionE.colilipopolysaccharide(LPS)suppliedbyACCisaUSPcertifiedcontrolstandardendotoxin(CSE)providedasalyophilizedpowder.Preparethestocksolutionof1000EU/mLbyreconstitutionoftheControl-StandardEndotoxin(CSE,E.coliO55:B5Endotoxin3).
Removethemetalsealfromthevial,breakthevacuumbyliftingthestopperjustenoughtoallowairtoenter,andasepticallyremovethestopper.AddLALReagentwaterdirectlyandwithcautiontotheCSEvial.ThefinalvolumeneededforreconstitutionoftheCSEvialshouldbecalculatedforeachlotanddependsonproductpotencydeterminedwithaspecificlotofLALreagentrelativetothecurrentFDAorUSPlotofreference.Thespecificvolumeneededtoreachapotencyof1000EU/mLcanbecalculatedfromtheCustomCertificateofAnalysis for theKineticTurbidimetricMicroplateMethod(providedbythesupplierAssociatesofCapeCod,Inc.3).
During reconstitution andprior to use, the stock solution should be vortexed vigorously for 30-60sec,with5-10minsettlingtimes,overa30-60mintimeframe,andallowedtoequilibratetoroomtemperature.VortextheCSEforatleast30secondseachtimeimmediatelybeforetakinganaliquotforusagetomakeappropriatedilutions.
ThereconstitutedstockCSEsolutionisstablefor4weeksstoredat2-8°C,donotfreezeCSE(ProductInsert CSE Endotoxin E. coli 0113:H10, ACC) 3. Before usage of the stored stock bring to room
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 7/14
temperatureandmixvigorouslyfor15minutesinordertoreleaseendotoxinthattendstoattachtotheglasssurface.
5.3.3 PreparationofEndotoxincalibrationstandardsIf the Pyrotell-T LAL Endotoxin Detection System (Associates of Cape Cod, Inc.) is being used in amicroplatereaderthedetectionlimit,andthusthelowestpossiblevalueofλis0.005EU/mL3.Adjustthe quantitative range of the assay and the sensitivity of an individual test defined by the lowestendotoxinconcentrationusedtoconstructthestandardcurve.
Labeldisposablepyrogen-freeglassdilutiontubesfortheendotoxindilutions.Prepareaseriousofendotoxinstandard-dilutionsbyadding0.1mLofthepriorendotoxinsolutioninto0.9mLofLALReagentWater.Eachdilutionshouldbevigorouslyvortexedforatleast1minutebeforeproceedingwiththenextstepofthedilutionseries.
Theendotoxincalibrationstandardsmaybepreparedasdescribedinthefollowingtable(alternativedilutionschemesmaybeused):
Dilutionschemeforpreparationofaseriesofendotoxinstandarddilutions
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A* 50* 100µLStockCSE+1900µLLALreagentwater
Cal.1 5.0100µLofInt.Asolution+900µLLALreagentwater
Cal.2 0.5 100µLCal.1+900µLLALreagentwater
Cal.3 0.05 100µLCal.2+900µLLALreagentwater
Cal.4 0.005 100µLCal.3+900µLLALreagentwater
*Thisisanexample;dilutionoftheCSEStocktomakeInt.AsolutionsdependsontheconcentrationofCSEstockandisdeterminedforeachlotofCSEreagent,refertotheCustomCertificateOfAnalysis.NumbersshowninthetableabovearecalculatedbasedonaStockconcentrationof1000EU/mL.
Eachsampleshouldbevigorouslyvortexedforatleastoneminutepriortouse.
5.4 AssayControlReactions
5.4.1 PreparationofInhibition/EnhancementtestusingaPositiveProductControl(PPC)Fortheverificationofresultsitisnecessarytopreparesampleswithadefinedamountofendotoxinstandardtodetermineinhibitionprocessesorinterferenceswiththeproduct.ThenominalendotoxinconcentrationspikedinIECshouldequalthatofastandarddilutionfromthemiddleofthestandard
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 8/14
curve and should be the same as used in theQuality Controls. For example, 25 µL of a 10EU/mLEndotoxinstandarddilutionareaddedto475µLnanoparticlesuspensionofthesample,resultingina spiked endotoxin concentration of 0.5 EU/mL. For the Quality control, the same amount ofEndotoxinisdilutedinto475µLLALreagentwater.
Dilutionscheme:PreparationofPositiveProductControls(PPC)
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A** 100.0* 100µLStockCSE+900µLLALreagentwater
Int.B** 10* 100µLofInt.A+900µLLALreagentwater
IEC 0.5 25µLofInt.B+475µLnanoparticlesuspension***
* Numbers shown in the tableaboveare calculatedbasedona Stock concentrationof1000EU/mL.** IntermediatesolutionsA,B arepreparedonlytomakecontroldilutionsandarenotused inassay.*** Theconcentrationofnanoparticles in IECshouldbeequal tooneassayed for standardcurve.YouwillneedtoprepareanIECforeachdilutionofthenanomaterialassayedinthistest.
Eachsampleshouldbevigorouslyvortexedforat leastoneminutepriortouse.Transfer100μLoftheIECsolutionintothe96-wellplateasdirectedbytheassaytemplate.
5.4.2 QualityControlsDilutionscheme:PreparationofQualityControls
SampleNominalConcentration
(EU/mL)PreparationProcedure
Int.A** 100.0* 100µLStockCSE+900µLLALreagentwater
Int.B** 10.0* 100µLofInt.A+900µLLALreagentwater
QC 0.5 25µLofInt.B+475µLLALreagentwater
* Numbers shown in the tableaboveare calculatedbasedona Stock concentrationof1000EU/mL.** IntermediatesolutionsA,B arepreparedonlytomakecontroldilutionsandarenotused inassay.*** The nominal concentration in QC should equal that of a standard from the middle of thestandardcurveandshouldbethesameasinIEC.
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 9/14
Eachsampleshouldbevigorouslyvortexedforat leastoneminutepriortouse.Transfer100μLoftheQCsolutionintothe96-wellplateasdirectedbytheassaytemplate.
5.4.3 NegativeControlUseendotoxinfreeLALreagentwaterorrespectivediluentbufferforthesamplesasanegativecontrolreference.
Transfer100μLofeachpreparedAssayControlReactionintothe96-wellplateasdirectedbytheassaytemplate.
6 Procedure
6.1 GeneralremarksMostimportantlymicrobialorendotoxincontaminationofallsamplesandmaterialshavingcontactwiththesampleandallusedtestreagentsmustbeavoidedbycarefulhandlingandtechnique.
6.2 PreparationofStudySamplesStudysamplesshouldbereconstitutedineitherLALreagentwaterorsterile,pyrogen-freePBS.ThepHof thestudysampleshouldbechecked. Itmaybenecessary toadjust thepHof thesample towithin therange6.0–8.0usingeithersterileendotoxin-freesodiumhydroxideorhydrochloricacid.DonotadjustthepHofunbufferedsolutions.Pyrogen-freeTrisbuffermayalsobeusedtopreparesamplesforendotoxindetectioninplaceofwaterasasamplediluenttoadjustpHofhighlyacidicorbasicsamples.ToavoidsamplecontaminationalwaysmeasurethepHofanaliquotofthepreparedsample. If the samplewas prepared in PBS or other diluent, the diluent alonemust be tested forendotoxin contamination in the assay. The concentration of nanomaterial is unique to eachformulation.Thegoalofthistestistomeasureendotoxinlevelpermgofthetestformulation,whichcommonlyreferstotheactivepharmaceutical ingredient(API),butmayalsobemeasuredinmgoftotalformulationortotalelement(e.g.goldorsilver).ThesampleshouldtestedfromthestockusingseveraldilutionsnotexceedingsocalledMaximumValidDilution(MVD).
To determine the MVD one needs to know three parameters: endotoxin limit (EL), sampleconcentrationandassaysensitivity(λ).ELiscalculatedaccordingtothefollowingformula:EL=K/M,whereKismaximumendotoxinlevelallowedperdose(5EU/kgforallroutesofadministrationexceptfortheintrathecalroute,forwhichKis0.2EU/kg)andMisthemaximumdosetobeadministeredper kg of body weight per single hour (1). Note, estimation of EL for nanomaterials used asradiopharmaceuticalorasmedicaldevicewillbedifferent5.Whenthedoseinformationforthetestnanomaterial isavailablebasedonananimalmodel(e.g. inmouse),onemayuseittoconvertintohumanequivalentdose(HED).Todosotheanimaldoseisdividedbytheconversionfactorspecifictoeachanimalspecies,e.g.12.3formouse.Pleaserefertoguidelinesforotherconversionratios6.Doseforcancertherapeutics isoftenprovidedinmg/m2insteadofmg/kg.Toconvertananimalorhuman dose frommg/m2 to mg/kg the dose in mg/kg is divided by the conversion factor of 37,indicatedaskm(formassconstant).Thekmfactorhasunitsofkg/m2;itisequaltothebodyweightinkgdividedbythesurfaceareainm2.Example74mg/m2/37=2mg/kg6.
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 10/14
TheMVDisdeterminedaccordingtothefollowingformula:MVD=(ELxsampleconcentration)/λ).Forexample,whennanoparticlesampleconcentrationis10mg/mLandit’smaximumdoseinmouseis 123 mg/kg, the HED is 123/12.3=10mg/kg; EL for all routes except intrathecal is 0.5 EU/mg(5EU/kg/10mg/kg) and MVD is 1000 ((0.5 EU/mg x 10 mg/mL)/0.005EU/mL). In this case, thenanomaterial will be tested directly from stock or at several dilutions not exceeding theMVD of1000,e.g. 10,100and1000timesdilution.Whentheinformationaboutthedoseisunknown,thehighestfinalconcentrationofthetestnanomaterialis1mg/mL,whichisusedtocalculatetheMVD.It is very important to recognize that if the dose, route of administration and/or the sampleconcentrationforthetestnanomaterialchange,theELandMVDwillalsochange.
6.3 Flowchart
Figure1:Briefoutlineoftheworkflow.
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6.4 MeasurementProcedure
6.4.1 TestprocedureTurnonthereaderinstrumentapproximately20-30minutesbeforestartingtheassaytoallowtheinstrumenttowarmup.Settemperatureto37°C.Switchdetectionwavelengthto340nm,andadjustallsettingsaslistedbelow,oruseapreinstalledprogramwhichisaccordingtothesesettings.
SettingforautomatedtemperaturecontrolledMicroplatereader:
- 340nmabsorbance- 37°Cincubationtemperature- 160–180cycleswith45scycletime
(differentcyclemaybeusedwithtotalmeasurementtime:approximately2h)
Alternatively,differentcycletimeswithadaptednumberofperformedcyclesmaybeusedwithatotalreadingtimeofatleast7200secondstoallowtimeforsampleswithlowamountsofendotoxintodevelop.Note:somelotsofthelysatearelesssensitivethanothers,ifthesensitivityoftheparticularlotislow,thetotalmeasurementtimemayneedtobeadjustedto9000sorlongerinordertoallowthelowestcalibratortodevelop.
6.4.2 Preparationofthe96-wellmicroplateDispense100µLofpreparedendotoxinstandards,differentproductsampledilutions(S),productinhibitionsamples(PPC),Qualitycontrol(QC),andblankEndotoxinfreeLALreagentwaterordiluentbuffer(BW)inthewellsofthe96wellplate.Prepareeachsampleatleastinduplicates.
Thefollowingmatrixcanbeusedasanexampletemplateforpipettingofanassaydesignedfor3test-samplesinthreedifferentdilutions,eachwithaPCC,allreactionsperformedinduplicates.Otherplatedesignsmaybeadvantageoustoapplyfordifferentsampleandcontrolreactionnumbers.
1 2 3 4 5 6 7 8 9 10 11 12
5 0.5 0.05 0.005 S1.1 S1.1 S1.2 S1.2 S1.3 S1.3
5 0.5 0.05 0.005 S1.1PPC
S1.1PPC
S1.2PPC
S1.2PPC
S1.3PCC
S1.3PCC
OC QC BW BW S2.1 S2.1 S2.2 S2.2 S2.3 S2.3
S2.1PPC
S2.1PPC
S2.2PPC
S2.2PPC
S2.3PCC
S2.3PCC
S3.1 S3.1 S3.2 S3.2 S3.3 S3.3
DocumentType DocumentID Version Status PageSOP EUNCL-STE-001.2.2 1.1 12/14
S3.1PPC
S3.1PPC
S3.2PPC
S3.2PPC
S3.3PCC
S3.3PCC
Shortly before usage reconstitute the necessary LAL Reagent vials with LAL Reagent Water orGlucashield®buffer(accordingtothemanufacturersinstruction),mixonlygently(donotvortex!)asdescribedabove(fordetailsreadunder-ReagentPreparation-).
Use a dispenser to immediately add 100 µL reconstituted LAL- Reagent into each of the reactionwells.Workquicklybutcarefultoavoidcausingbubblesinthewell.Controlallwellsforabsentsofbubbles.
Mix gently for about 15 seconds and start automated reading procedure immediately (reading isperformedwiththemicroplatecoverremoved).
Duringthemeasurementtime,donotdisturbthereactionplate.Thelaboratorybenchsupportingtheopticalreadershouldbefreefromexcessivevibration.
6.5 DataAnalysisThereactiontimeneededfortheappearanceofturbidityisinverselyproportionaltotheamountofpresentendotoxin.Forthedeterminationoftheexactendotoxinamountitisnecessarytocreateastandard curve of at least 3 to 4 different concentrations (e. g. 5 EU/mL, 0.5 EU/mL, 0.05 EU/mL,0.005EU/mL).Basedon the values for theendotoxin standard curvea log/log linear correlation isusedtocalculatevaluesof thecorrespondingEndotoxinconcentration inEU/mLfromthereactiontime. The initial absorbance of each well is used as blank for its subsequent kinetic readings toperformabaselinecorrectionandtodeterminethetimetoreachan increaseof0.100absorbanceunits.Thecorrelationcoefficientabsolutevalueforthestandardcurveshouldbe≥0.980toenableareliable interpolationofunknownsamples. Thedifferentparametersare theabsorptionvalues forthex-range,meanreactiontimeforthey-range,and0.100asthresholdvalue,inordertodeterminethereactiontimefortheincreaseof0.1absorbanceunits.Constructastandardcurvebyregressionofthelogonsettimeagainstthelogendotoxinconcentrationforthestandards.Theequationfortheregression line describes the standard curve. The line equation of the standard curve is used forcalculationofendotoxinconcentrationsofthesamples(includingstandardsandcontrols).Analysisisperformed by the appropriate template of theMARS Data Analysis Software (BMG Labtech). Thesoftware is used to directly calculate the results from the microplate reader of the kineticturbidimetricLALassay.
Therecoveryrateofpositiveproductcontrol(PPC)andQualitycontrols(QC)iscalculatedbydividingthe measured spiked endotoxin concentration by the nominated one to determine potentialinhibitionorenhancementreactionsofthesampleingredientsattherespectiveconcentrationofthetestedsample.
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7 AssayAcceptanceCriteria1. Linearregressionalgorithmisusedtoconstructthestandardcurve.Precision(%CV)and
accuracy(PDFT)ofeachcalibrationstandardandqualitycontrolshouldbewithin25%.2. Atleastthreecalibrationstandardsshouldbeavailableforassaytobeconsidered
acceptable.3. Thecorrelationcoefficientofthestandardcurvemustbeatleast0.980.4. Ifqualitycontrolsfailtomeetacceptancecriteriondescribedin7.1,runshouldberepeated.5. Ifstandardcurvefailstomeetacceptancecriteriondescribedin7.1–7.3,therunshouldbe
repeated.6. Precisionofthestudysampleshouldbewithin25%.7. Precisionofinhibition/enhancementcontrolshouldbewithin25%.8. Spikerecoveryindicativeoftheaccuracyoftheinhibition/enhancementcontrolshould
between50and200%[4].Spikerecoverylessthan50%isindicativeofinhibition;thatabove200%isindicativeofeitherendotoxincontaminationorenhancement.
9. Ifsampleinterferenceisdetected,theassayresultsforthissampleareinvalid.Othertestsshouldbeconsideredasdiscussedinreference5.
8 HealthandSafetyWarnings,CautionsandWasteTreatmentUsePyrotell-Tforinvitrodiagnosticpurposesonly.Donotuseitforthedetectionofendotoxemia.Thetoxicityofthereagenthasnotbeendetermined;thus,cautionshouldbeexercisedwhenhandlingPyrotell-T.
Informyourselfaboutthecontentandsamplematerialandallrelevantsafetyissuesconcerningthesamplesbeforeunpackingandhandlingofanyreceivedsample.
Alwayswearadequatepersonalprotectiveequipment,inparticularprotectivelaboratorycoat,glovesandsafetyglassesandtakeallnecessaryprecautionstoprotectyourselfandothers.Glovesmustbeinspectedpriortouse.Usepropergloveremovaltechnique(withouttouchingglove'soutersurface)toavoidskincontactwiththeproducts.Userespiratoryprotectionwheneveradvisable.Openthesamplevialsonlyundersterileconditionsofthesterileworkbenchinordertoavoidsamplespillingandcontamination.Takeallnecessaryprecautionstoavoidanyfurthersamplespillingincaseofdamagedsamplecontainer.Wastedisposalhastobeproceededinaproperformusingmeansadequateforthematerialspecifications,incompliancewiththelaboratoryregulationsandgeneralregulatoryconditionsaccordingtoapplicablelegalregulations.
9 AbbreviationsAPI activepharmaceuticalingredient
BW blankwater
CV coefficientofvariation
EL EndotoxinLimit
EU endotoxinunit
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HCl hydrochloricacid
LAL LimulusAmebocyteLysate
MVD MaximumValidDilution
NaOH sodiumhydroxide
PBS phosphatebufferedsaline
PES polyethersulfone
PPC positiveproductcontrol
RT roomtemperature
S sample
10 References1:Bang,F.B.AbacteriladiseaseofLimuluspolyphemus.Bull.JohnsHopkinsHosp.98:325(1956)
2:Levin,J.,Bang.F.B.ClottableproteininLimulus:itslocalisationandkineticsofitscoagulationbyendotoxin.Thromb.Diath.Haemorrh.19:186(1968)
3:USP34-NF29.<85>.BacterialEndotoxins.Rockville,MD:UnitedStatesPharmacopeia,2011,Volume1,78-81.
4:FDAGuidanceforIndustryandReviewersEstimatingtheSafeStartingDoseinClinicalTrialsforTherapeuticsinAdultHealthyVolunteers.December2002.
5:USFDA.GuidanceforIndustry.PyrogenandEndotoxinstesting:Questionsandanswers,2012.