最新整理细菌内毒素USP教学提纲Word下载.docx

最新整理细菌内毒素USP教学提纲Word下载.docx

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Portionsofthisgeneralchapterhavebeenharmonizedwiththecorrespondingtextsofthe 

EuropeanPharmacopoeia 

and/orthe 

JapanesePharmacopoeia.Thoseportionsthatarenotharmonizedaremarkedwithsymbols（

）tospecifythisfact.

这一章节已经和欧洲药典（EP）与日本药典（JP）统一。

没有统一的部分已经用

标记出来。

TheBacterialEndotoxinsTest（BET）isatesttodetectorquantifyendotoxinsfromGram-negativebacteriausingamoebocytelysatefromthehorseshoecrab（Limuluspolyphemus 

or 

Tachypleustridentatus）.

细菌内毒素检查法（BET）是通过鲎的阿米巴细胞溶解产物来检测或定量来自革兰氏阴性菌的内毒素。

Therearethreetechniquesforthistest:

thegel-clottechnique,whichisbasedongelformation;

theturbidimetrictechnique,basedonthedevelopmentofturbidityaftercleavageofanendogenoussubstrate;

andthechromogenictechnique,basedonthedevelopmentofcoloraftercleavageofasyntheticpeptide-chromogencomplex.Proceedbyanyofthethreetechniquesforthetest.Intheeventofdoubtordispute,thefinaldecisionismadebaseduponthegel-clottechniqueunlessotherwiseindicatedinthemonographfortheproductbeingtested.Thetestiscarriedoutinamannerthatavoidsendotoxincontamination.

细菌内毒素检查法有3种：

凝胶法，基于凝胶的形成；

浊度法，

BACTERIALENDOTOXINSTESTUSP32

PortionsofthisgeneralchapterhavebeenharmonizedwiththecorrespondingtextsoftheEuropeanPharmacopeiaand/ortheJapanesePharmacopeia.Thoseportionsthatarenotharmonizedaremarkedwithsymbols（

Thischapterprovidesatesttodetectorquantifybacterialendotoxinsthatmaybepresentinoronthesampleofthearticle（s）towhichthetestisapplied.ItusesLimulusAmebocyteLysate（LAL）obtainedfromtheaqueousextractsofcirculatingamebocytesofhorseshoecrab（Limuluspolyphemus 

Tachypleustridentatus）whichhasbeenpreparedandcharacterizedforuseasanLALReagent.

1

Therearetwotypesoftechniquesforthistest:

thegel-clottechniques,whicharebasedongelformation,andthephotometrictechniques.Thelatterincludeaturbidimetricmethod,whichisbasedonthedevelopmentofturbidityaftercleavageofanendogenoussubstrate,andachromogenicmethod,whichisbasedonthedevelopmentofcoloraftercleavageofasyntheticpeptide-chromogencomplex.Proceedbyanyoneofthesetechniques,unlessotherwiseindicatedinthemonograph.Incaseofdispute,thefinaldecisionisbasedonthegel-clottechniques,unlessotherwiseindicatedinthemonograph.

Inthegel-clottechniques,thereactionendpointisdeterminedfromdilutionsofthematerialundertestindirectcomparisonwithparalleldilutionsofareferenceendotoxin,andquantitiesofendotoxinareexpressedinUSPEndotoxinUnits（USP-EU）. 

[NOTE—OneUSP-EUisequaltooneIUofendotoxin.]

BecauseLALReagentshavebeenformulatedtobeusedalsoforturbidimetricorcolorimetrictests,suchtestsmaybeusedtocomplywiththerequirements.Thesetestsrequiretheestablishmentofastandardregressioncurve;

theendotoxincontentofthetestmaterialisdeterminedbyinterpolationfromthecurve.TheproceduresincludeincubationforapreselectedtimeofreactingendotoxinandcontrolsolutionswithLALReagentandreadingofthespectrophotometriclightabsorbanceatsuitablewavelengths.Intheendpointturbidimetricprocedurethereadingismadeimmediatelyattheendoftheincubationperiod.Intheendpointcolorimetricprocedurethereactionisarrestedattheendofthepreselectedtimebytheadditionofanenzymereaction-terminatingagentpriortothereadings.Intheturbidimetricandcolorimetrickineticassaystheabsorbanceismeasuredthroughoutthereactionperiodandratevaluesaredeterminedfromthosereadings.

APPARATUSANDGLASSWARE

Depyrogenateallglasswareandotherheat-stablematerialsinahot-airovenusingavalidatedprocess.

2

Commonlyusedminimumtimeandtemperaturesettingsare30minutesat250

.Ifemployingplasticapparatus,suchasmicroplatesandpipettipsforautomaticpipetters,useonlythatwhichhasbeenshowntobefreeofdetectableendotoxinandnottointerferewiththetest. 

[NOTE—Inthischapter,theterm“tube”includesanyotherreceptaclesuchasamicro-titerwell.]

PREPARATIONOFTHESTANDARDENDOTOXINSTOCKSOLUTIONANDSTANDARDSOLUTIONS

The 

USPEndotoxinRS 

hasadefinedpotencyof10,000USPEndotoxinUnits（EU）pervial.Constitutetheentirecontentsof1vialoftheRSEwith5mLofLALReagentWater

3

mixintermittentlyfor30minutes,usingavortexmixer,andusethisconcentrateformakingappropriateserialdilutions.Preservetheconcentrateinarefrigeratorformakingsubsequentdilutionsfornotmorethan14days.Mixvigorously,usingavortexmixer,fornotlessthan3minutesbeforeuse.Mixeachdilutionfornotlessthan30secondsbeforeproceedingtomakethenextdilution.Donotstoredilutions,becauseof