EP8.0细菌内毒素检查法中英文对照

EP8.02.6.14细菌内毒素（中英文）2.6.14.BACTERIALENDOTOXINS细菌内毒素Thetestforbacterialendotoxins(BET)isusedtodetectorquantifyendotoxinsfromgram-negativebacteriausingamoebocytelysatefromthehorseshoecrab(LimuluspolyphenmusorTachypleustridentatus).Thereare3techniquesforthistest:thegel-clottechniques,whichisbasedongelformation;theturbidimetrictechnique,basedonthedevelopmentofturbidityaftercleavageofanendogenousesubstrate;andthechromogenictechnique,basedonthedevelopmentofcolouraftercleavageofasyntheticpeptide-chromogencomplex.本法利用鲎试剂（从鲎——美洲鲎或中国鲎——变形细胞溶解物制备而来）检测由革兰氏阴性菌产生的细菌内毒素或对内毒素进行定量。该检查包括三种方法：一为凝胶法，系利用鲎试剂与内毒素产生凝集反应的原理；第二种为浊度法（基于内源性底物断裂后，产生的浊度变化）；最后一种为显色法（得到的肽－呈色基团复合物断裂后，检测反应混合物的色度）。Thefollowing6methodsaredescribedinthepresentchapter:这一章阐述了下面6种方法：MethodA.Gel-clotmethod:limittestMethodB.Gel-clotmethod:quantitativetestMethodC.TurbidimetrickineticmethodMethodD.ChromogenickineticmethodMethodE.Chromogenicend-pointmethodMethodF.Turbidimetricend-pointmethod方法A：凝胶法：限度试验方法B：凝胶法：定量试验方法C：动态浊度法方法D：动态显色法方法E：终点显色法方法F：终点浊度法Proceedbyanyofthe6methodsforthetest.Intheeventofdoubtordispute,thefinaldecisionismadebaseduponmethodAunlessotherwiseindicatedinthemonograph.检测时，可用6种方法的任一种进行试验。当测定结果可疑或有争议时，除非另有规定，以专论中的方法A的测定结果为准。Thetestiscarriedoutinamannerthatavoidsendotoxincontamination.试验操作过程应防止内毒素的污染。1.APPARATUS仪器Depyrogenateallglasswareandotherheat-stableapparatusinahot-airovenusingavalidatedprocess.Acommonlyusedminimumtimeandtemperatureis30minutesat25?C.Ifemployingplasticapparatus,suchasmicrotitreplatesandpipettetipsforautomaticpipetters,useapparatusshowntobefreeofdetectableendotoxinandwhichdoesnotinterfereinthetest.所有的玻璃器皿及由其他耐热材料制成的器皿需用已验证的工艺在热烘箱内进行去热原处理。去热原时，常用的最小时间和温度设置分别为30分钟和250℃。若使用塑料器械，如微孔板和微量进样器配套的吸头等，它们必须标明无内毒素并确对试验无干扰。NOTE:inthischapter,theterm‘tube’includesalltypesofreceptacles,forexamplemicrotitreplatewells.注：这一章中，“管”的意思包括其他任何反应容器，如微孔板中的孔。2.REAGENTS,TESTSOLUTIONS试剂、试液(1)Amoeboytelysate鲎试剂Amoebocytelysateisalyophilizedproductobtainedfromamoebocytelysatefromthehorseshoecrab(LimuluspolyphemusorTachypleustridentatus).Thisreagentrefersonlytoaproductmanufacturedinaccordancewiththeregulationsofthecompetentauthority.鲎试剂物是从鲎（美洲鲎或中国鲎）的变形细胞产生的低压冻干产物。该试剂仅指在符合有关权威法规的生产条件下生产的鲎试剂产品。NOTE:amoebocytelysatereactswithsomeβ-glucansinadditiontoendotoxins.Amoebocytelysatepreparationswhichdonotreactowithglucansareavailable;theyarepreparedbyremovingfromamoebocytelysatetheGfactor,whichreactswithglucans,orbyinhibitingtheGfactorreactoingsystemofamoebocytelysate.Thesepreparationsmaybeusedforendotoxintestinginthepresenceofglucans.注：除了内毒素外，鲎试剂和β-葡聚糖反应。也存在不与葡聚糖反应的鲎试剂；它们通过从变形细胞溶解物中除去G因子来制备，因为G因子会和葡聚糖反应，或者通过抑制变形细胞溶解物的G因子反应系统来制备。这些制剂可被用于葡聚糖存在情况下的内毒素测试。(2)Lysatesolution鲎试液DissolveamoebocytelysateinwaterforBETorinabuffer,asrecommendedbythelysatemanufacturer,bygentlestirring.Storethereconstitutedlysate,refrigeratedorfrozen,asindicatedbythemanufacturer.通过缓慢搅拌，将鲎试剂溶于水或溶于厂家推荐的缓冲液中来进行细菌内毒素试验（BET）。按照厂商的指示，冷藏或冷冻储存重新鲎试液。(3)WaterforBET(waterforbacterialendotoxinstest)BET用水WaterforinjectionsRorwaterproducedbyotherproceduresthatshowsnoreactionwiththelysateemployedatthedetectionlimitofthereagent.BET用水指注射用水R或由其他方法制取的内毒素含量低于所用鲎试剂的检测限的水。3.PREPARATIONOFTHESTANDARDENDOTOXINSTOCKSOLUTION（内毒素储备标准溶液的制备）ThestandardendotoxinstocksolutionispreparedfromanendotoxinreferencestandardthathasbeencalibratedagainsttheInternationalStandard,forexampleendotoxinstandardBRP.用内毒素标准品制备内毒素储备标准溶液；所用的内毒素标准品必须先用国际标准品校准，如内毒素标准BRP。EndotoxinisexpressedinInternationalUnits(IU).TheequivalenceinIUoftheInternationalStandardisstatedbyWorldHealthOrganisation.内毒素以国际单位（IU）表示。IU的换算见国际卫生组织（WHO）公布的国际标准。NOTE:oneInternationalUnit(IU)ofendotoxinisequaltooneEndotoxinUnit(E.U.).注：一国际单位（IU）内毒素相当于一个内毒素单位（E.U.）。Followthespecificationsinthepackageleafletandonthelableforpreparationandstorageofthestandardendotoxinstocksolution.根据包装说明书上的标准和内毒素储备标准溶液的标签上关于制备和贮存的说明。4.PREPARATIONOFTHESTANDARDENDOTOXINSOLUTIONS（内毒素标准溶液的制备）Aftervigorouslymixingthestandardendotoxinstocksolution,prepareappropriateserialdilutionsofthissolutionusingwaterforBET.充分混合内毒素储备标准溶液后，用细菌内毒素试验检查用水（BET用水）稀释，制成适当的系列稀释液，即得BET用内毒素标准溶液。Usethesolutionassoonaspossibletoavoidlossofactivitybyadsorption.得到的稀释液应尽快使用，以免因吸附而导致活性损失。5.PREPARATIONOFTHETESTSOLUTIONS供试品溶液的制备PreparethetestsolutionsbydissolvingordilutingactivesubstancesormedicinalproductsusingwaterforBET.Somesubstancesorpreparationsmaybemoreappropriatelydissolvedordilutedinotheraqueoussolutions.Ifnecessary,adjustthepHofthetestsolution(ordilutionthereof)sothatthepHofthemixtureofthelysateandtestsolutionfallswithinthepHrangespecifiedbythelysatemanufacturer,usually6.0to8.0.thepHmaybeadjustedbytheuseofacid,baseorasuitablebuffer,asrecommendedbythelysatemanufacturer.Acidsandbasesmaybepreparedfromconcentratesorsol8idswithwaterforBETincontainersfreeofdetectableendotoxin.Buffersmustbevalidatedtobefreeofdetectableendotoxinandinterferingfactors.除非另有说明，以BET用水溶解或稀释活性成分或药品来制备供试品溶液。某些成分或药品可能在其它的水溶液中溶解度更高。如有必要，调节待测溶液（或它的稀释液）的pH值，使鲎试剂和供试品溶液的混合物的pH值在所选鲎试剂生产商的指定pH范围内。一般要求供试品溶液的pH值范围为6.0——8.0。可用鲎试剂生产商推荐的酸、碱或适当的缓冲溶液来调解pH值。酸或碱溶液须用BET检查用水在去除内毒素的容器中配制。