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Dissolveamoebocytelysateinwater...