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1、REAGENTSANDTESTSOLUTIONS试剂和测试溶液AmoebocyteLysate-Alyophilizedproductobtainedfromthelysateofamoebocytes(whitebloodcells)fromthehorseshoecrab(LiniuluspolyphemusorTachypleustridentatus).Thisreagentrefersonlytoaproductmanufacturedinaccordancewiththeregulationsofthecompetentauthority.oiE-AmoebocyteLysater
2、eactstosomeB-glucansinadditiontoendotoxins.AmoebocyteLysatepreparationsthatdonotreacttoglucansareavailable:theyarepreparedbyremovingtheGfactorreactingtoglucansfromAmoebocyteLysateorbyinhibitingtheGfactorreactingsystemofAmoebocyteLysateandmaybeusedforendotoxintestinginthepresenceofglucans.变形细胞溶解液一由赏的
3、(白血细胞)变形细胞溶解液制得的冻干产品。该试剂仅指那些按照相关的监管机构的法规要求制得的产品。注：变形细胞溶解液除了与内毒素反响外，还会与某些B-葡聚糖反响。可以制成不与B-葡聚糖发生反响的变形细胞溶解液制品：可通过从变形细胞溶解液中去除会与葡聚糖反响的G因子或抑制变形细胞溶解液的G因子反响系统来制得该制品，这些制品可用于在存在葡聚糖时进行内毒素检测。WaterforBacterialEndotoxinsTest(BET)一UseWaterforInjectionorwaterproducedbyotherproceduresthatshowsnoreactionwiththelysatee
4、mployed,atthedetectionlimitofthereagent.细菌内毒素检测(BET)用水一使用注射用水或用其他方式制得的不会与赏试剂检测限对应浓度的溶菌液发生反响的水。1.ysateTS一DissolveAmoebocyteLysateinWaterforBET,orinabufferrecommendedbythelysatemanufacturer,bygentlestirring.Storethereconstitutedlysate,refrigeratedorfrozen,accordingtothespecificationsofthemanufacturer.
5、赏试剂一在缓慢搅拌的同时，将变形细胞溶解液溶于细菌内毒素检测用水中或溶于溶菌液生产商推荐的缓冲液中。按照生产商的说明来冷藏或冷冻储存该再生溶菌液。GEL-CLOTTECHNIQUE凝胶法TheGel-ClotTechniqueisfordetectingorquantifyingendotoxinsbasedonclottingofthelysatereagentinthepresenceofendotoxin.Theminimumconcentrationofendotoxinrequiredtocausethelysatetoclotunderstandardconditionsisthe
6、labeledsensitivityofthelysatereagent.Toensureboththeprecisionandvalidityofthetest,performthetestsforconfirmingthelabeledlysatesensitivityandforinterferingfactorsasdescribedunderPreparatoryTesting.凝胶法通过赏试剂与内毒素产生凝集反响的原理，来检测或定量内毒素。在标准条件下使溶菌液凝结所需的最低内毒素浓度即为赏试剂的标示灵敏度。为了确保化验的精密度和有效性，按照凝咬注预各岚驳项所述来确认量试剂标示灵敏度
7、和干扰因子。PreparatoryTesting凝胶法预备试验TestforConfirmationofLabeledLysateSensitivityConfirminfourreplicatesthelabeledsensitivity,expressedinEU/mLofthelysatepriortouseinthetest.Thetestforconfirmationoflysatesensitivityistobecarriedoutwhenanewbatchoflysateisusedorwhenthereisanychangeinthetestconditionsthatmay
8、affecttheoutcomeofthetest.Preparestandardsolutionshavingatleastfourconcentrationsequivalentto2,bydilutingtheUSPEndotoxinRSwithWaterforBET.确认匿试剂标示灵敏度一在进行化验之前，要对臂试剂标示灵敏度入(Eml)重复确认4次。当使用新批次的邕试剂或当实验条件出现任何可能影响实验结果的变更时，均应进行邕试剂灵敏度确实认。用细菌内毒素检测用水瞬释内毒素USP标准品，配制一系列标准溶液，至少含有与2入，入，和人相当的四种浓度。MixavolumeoftheLysate
9、TSwithanequalvolume(suchas0.1-mLaliquots)ofoneoftheStandardEndotoxinSolutionsineachtesttube.Whensingletestvialsorampulscontaininglyophilizedlysateareused,addsolutionsdirectlytothevialorampul.Incubatethereactionmixtureforaconstantperiodaccordingtothedirectionsofthelysatemanufacturer（usuallyat371for60
10、+2minutes）,avoidingvibration.Totesttheintegrityofthegel,takeeachtubeinturndirectlyfromtheincubatorandinvertitthroughabout1800inonesmoothmotion.Ifafirmgelhasformedthatremainsinplaceuponinversion,recordtheresultaspositive.Aresultisnegativeifanintactgelisnotformed.Thetestisconsideredvalidwhenthelowestc
11、oncentrationofthestandardsolutionsshowsanegativeresultinallreplicatetests.在每个试管中将一定体积的董岚宛与等体积（例如0.1mL的等份）的其中一种标准的毒黄港滋进行混合。当使用含有冻干邕试剂的一次性实验瓶或者安甑时，向瓶或安甑中直接参加溶液。按照赏试剂生产商的规定，要在恒定时段内将反响混合物保持恒温（通常是371条件下602分钟），防止振动。为检验凝胶体的完整性，依次直接从恒温箱中取出各个试管，并平滑地将其翻转180。如果已经形成的牢固凝胶在倒置后保持原位，那么将结果记录为阳性。如果没有形成完整的凝胶，那么结果为阴性。只
12、有当最低浓度的标准溶液在所有重复试验中均呈阴性时，该试验方为有效。Theendpointisthesmallestconcentrationintheseriesofdecreasingconcentrationsofstandardendotoxinthatclotsthelysate.Determinethegeometricmeanendpointbycalculatingthemeanofthelogarithmsoftheendpointconcentrationsofthefourreplicateseriesandthentakingtheantilogarithmoftheme
13、anvalue,asindicatedinthefollowingformula:反响终点就是引起溶菌液凝结的系列递减的标准内毒素浓度中的最低一个浓度。如下式所示，计算四次重复试验的系列终点浓度的对数平均值，然后计算该平均值的反对数，从而得到几何平均终点。GeometricMeanEndpointConcentration=antilog（Sef）几何平均终点浓度=Sef的反对数andnotmorethan2,thelabeledsensitivityisconfirmedandisusedintestsperformedwiththislysate.其中，Se指的是所使用的系列稀释液的终点浓
14、度的对数和，f是重复试验的次数。几何平均终点浓度即为赏试剂灵敏度的测定值（以EU/mL为单位)。如果该值不小于入且不大于2人，那么标示灵敏度得到了确认，并可用于使用此溶菌液进行的试验。TestforInterferingFactorsUsuallypreparesolutions(A-D)asshowninTable7,andperformtheinhibition/enhancementtestontheSampleSolutionsatadilutionlessthantheMVD,notcontaininganydetectableendotoxins,operatingasdescri
15、bedforTestforConfirmationofLabeledLysateSensitivity.ThegeometricmeanendpointconcentrationsofSolutionsBandCaredeterminedusingtheformuladescribedintheTestforConfirmationofLabeledLysateSensitivity.干扰因子检测：按照表1所示配制溶液A、溶液B、溶液C、溶液D,并且按照上述确实以蛰岚砌麻灾敏度项所述，对稀释倍数不超过最大有效稀释倍数(MVD),不含有任何内毒素的相名落滋进行抑制/增强试验。用癌以董斌砌玩示灵敏
16、度项中提及的公式来测定溶液8和落液C的几何平均终点浓度。Table1.PreparationofSolutionsfortheInhibitionZEnhancementTestforGel-ClotTechniques表1：用于凝胶法抑制/增强试验的溶液制备Solution溶液EndotoxinConcentration/SolutiontowhichEndotoxinisAdded内毒素浓度/参加内毒素的溶液Diluent稀释液DilutionFactor稀释因子EndotoxinConcentration内毒素浓度NumberofReplicates重复次数AaNone/SampleSo
17、lution无I样品溶液4BbikSampleSolution2入样品SampleSolution样品12人4溶液溶液21人入入44484G2WaterforBET2以细菌WaterforBET细菌内毒素12人2内毒素检测(BET)用水检测用水2l24282Solution溶液EndotoxinConcentration/SolutiontowhichEndotoxinisAdded内毒素浓度/参加内毒素的溶液Diluent稀释液DilutionFactor稀释因子EndotoxinConcentration内毒素浓度NumberofReplicates重复次数DaNone/WaterforB
18、ET天细菌内毒素检测用水2aSolutionA:ASampleSolutionofthepreparationundertestthatisfreeofdetectableendotoxins.溶液限用于化验的不含可检测到的内毒素的精陷落滋bSolutionB:Testforinterference.溶液B：干扰因子检测。cSolutionC:Controlforlabeledlysatesensitivity溶液C，.对邕试剂标示灵敏度的控制dSolutionD:NegativecontrolofWaterforBET溶液。.细菌内毒素检测用水用性可照、Thetestisconsidered
19、validwhenallreplicatesofSolutionsAandDshownoreactionandtheresultofSolutionCconfirmsthelabeledSenSitiVity.仅当溶液A和溶液D的所有重复试验均显示无反响，且洛滋C的结果确认标示灵敏度符合要求时，试验方为有效。IfthesensitivityofthelysatedeterminedinthepresenceofSolutionB入andnotgreaterthan2,theSampleSolutiondoesnotcontainfactorsthatinterfereundertheexper
20、imentalconditionsused.Otherwise,theSampleSolutiontobeexaminedinterfereswiththetest.如果用溶液B的供试品溶液测得入且不大于2人，那么该精就方法中在实验条件下不含有干扰因子。否那么，该样就溶正会干扰此试验。IfthesampleundertestdoesnotcomplywiththetestatadilutionlessthantheMVD,repeatthetestusingagreaterdilution,notexceedingtheMVD.Theuseofamoresensitivelysatepermi
21、tsagreaterdilutionofthesampletobeexaminedandthismaycontributetotheeliminationOfinterferenCe.如果供试品在稀释倍数小于最大有效稀释倍数(MVD)的情况下不符合该试验的要求，那么用一个更大的稀释倍数(但不超过MVD)来重复该试验。使用灵敏度更大的溶菌液允许对样品进行更大倍数的稀释，这样可以起到排除干扰的作用。Interferencemaybeovercomebysuitabletreatment,suchasfiltration,neutralization,dialysis,orheating.Toest
22、ablishthatthechosentreatmenteffectivelyeliminatesinterferencewithoutlossofendotoxins,performtheassaydescribedaboveusingthepreparationtobeexaminedtowhichUSPEndotoxinRShasbeenaddedandwhichhasthenbeensubmittedtothechosentreatment.可以通过适当的处理来克服干扰，例如过滤、中和、透析、或加热。为了确定所选用的处理方法能有效地排除干扰且不会造成内毒素的减少，要使用参加了内毒素US
23、a标准品并用所选方法进行了处理的供试品溶液，进行上述化验。1.imitTest限度检测ProcedurePrepareSolutionsA,B,C,andDasshowninTable2,andperformthetestonthesesolutionsfollowingtheprocedureforTestforConfirmationofLabeledLysateSensitivityunderPreparatoryTesting.程序一根据表2配制溶液鼠溶液B、溶液C、溶液0,并按照廉咬法预留试1卷中邕试剂标示灵敏度确实认项所述对这些溶液进行检测。Table 2. Preparation
24、ofSolutionsfortheGel-ClotLimitTest表2：凝胶限度检测，溶液的制备Solution-溶液2EndotoxinConcentration/SolutiontowhichEndotoxinisAdded内毒素浓度/参加内毒素的溶液NumberofReplicates重复次数ANoneZDilutedSampleSolution无/稀释的样名溶液2B2/DilutedSampleSolution2人/稀释的样品溶液2C2WaterforBETAJ细菌内毒素检测用水2DNOne/WserforBET无细菌内毒素检测用水2*PrepareSolutionAandthepo
25、sitiveproductcontrolSolutionBusingadilutionnotgreaterthantheMVDandtreatmentsasfortheTestforInterferingFactorsunderPreparatoryTesting.ThepositivecontrolSolutionsBandCcontaintheStandardEndotoxinSolutionataconcentrationcorrespondingtotwicethelabeledlysatesensitivity.ThenegativecontrolSolutionDconsistso
26、fWaterforBEZ使用不超过最大有效稀释倍数(MVD)的稀释度并按照凝咬注赖各试验中干批因子的检潮项规定的处理方法来配制溶液4和阳性产品对照落滋8。阳性对照溶滋B和C含有浓度为标示称邕试剂灵敏度两倍的标准内毒素溶液。阴性对照落彼D由细菌内毒素检测用水组成。Interpretation-ThetestisconsideredvalidwhenbothreplicatesofSolutionBandCarepositiveandthoseofSolutionDarenegative.WhenanegativeresultisfoundforbothreplicatesofSolutionA,
27、thepreparationundertestcomplieswiththetest.WhenapositiveresultisfoundforbothreplicatesofSolutionA,thepreparationundertestdoesnotcomplywiththetest.说明：只有当阳性对照落液B9C的两次重复试验均呈阳性，且阴性对照7期TD均呈阴性，此试验方为有效。假设溶液4的两次重复检测结果呈阴性，那么溶液配制符合试验要求。当溶液A的两次重复检测均呈阳性结果，那么供试品不符合此检测的要求。Whenapositiveresultisfoundforonereplicate
28、ofSolutionAandanegativeresultisfoundfortheother,repeatthetest.Intherepeattest,thepreparationundertestcomplieswiththetestifanegativeresultisfoundforbothreplicatesofSolutionA.ThepreparationdoesnotcomplywiththetestifapositiveresultisfoundforoneorbothreplicatesofSolutionA.However,ifthepreparationdoesnot
29、complywiththetestatadilutionlessthantheMVD,thetestmayberepeatedusingagreaterdilution,IloteXCeedingtheMVD.当溶液A的两次重复测试中，!个呈阳性结果，1个呈阴性结果时，须重复该实验。在重复该实验时，如果，期茯A均呈阴性结果，那么溶液配制符合此测试的要求。如果溶液4的一次或者两次的检测结果都是阳性,那么溶液配制不符合试验要求。但是，如果其稀释度小于最大有效稀释倍数(MVD)且溶液配制不符合实验要求，可以使用更大的稀释度来重复该实验，但该稀释度不得超过最大有效稀释倍数IMVD)。Quantitat
30、iveTest定量检测ProcedureThetestquantifiesbacterialendotoxinsinSampleSolutionsbytitrationtoanendpoint.PrepareSolutionsA,B,CandDasshowninTable3,andtestthesesolutionsbyfollowingtheprocedureintheTestforConfirmationofLabeledLysateSensitivityunderPreparatoryTesting.程序一通过滴定至终点来定量检测殍扇7新茯中的细菌内毒素。按照表3所示配制落族4溶液87辨
31、TC溶液。,并且按照凝芯法颜务试缰中螯试砌况裾敏度联以项所述来进行检测。Table 3. PreparationofSolutionsfortheGel-ClotAssay表3：凝胶法定量检测，溶液的制备Solution溶液EndotoxinConcentration/SolutiontowhichEndotoxinisAdded内毒素的含量/参加内毒素的溶液Diluent稀释液DilutionFaCtol稀释因子EndotoxinConcentration内毒素浓度NumberofReplicates重复次数Aa无/样品溶液WaterforBET12Solution溶液EndotoxinCo
32、ncentration/SolutiontowhichEndotoxinisAdded内毒素的含量/参加内毒素的溶液Diluent稀释液DilutionFaCtOr稀释因子EndotoxinConcentration内毒素浓度NumberofReplicates重复次数None/SampleSolution细菌内毒素检测用水248222Bb2SampleSolution2/样品溶液122G2WaterforBET2细菌内毒素检测用水WaterforBET细菌内毒素检测用水12482l2222DaNone/WoferforBET无I细菌内毒素检测用水2aSolutionA:SampleSolut
33、ionundertestatthedilution,nottoexceedtheMVD,withwhichtheTestforInterferingFactorswascompleted.SubsequentdilutionoftheSampleSolutionmustnotexceedtheMVD.UseWaterforBETtomakeadilutionseriesoffourtubescontainingtheSampleSolutionundertestatconcentrationsof1,and1/8relativetotheconcentrationusedintheTestfo
34、rInterferingFactors.OtherdilutionsuptotheMVDmaybeusedasappropriate.W:稀释度不超过最大有效稀释倍数(MVD)的供试品溶液，用于进行干扰因子检测0精曲溶液的后续稀释一定不得超过最大有效稀释倍数(MVD)o使细菌内毒素检测用水来酣制4管稀释液,其中样品落法浓度分别为干扰因子检测中用到的浓度的1、1/2、1/4、1/8倍。必要时也可以使用其他稀释度(不超过MVD)的稀释液。bSolutionB:SolutionAcontainingstandardendotoxinataconcentrationof2(positiveproduc
35、tcontrol）.:含有浓度为2入的内毒素标准品的溶液2（阳性控制）OcSolutionC:TworeplicatesoffourtubesofWaterforBETcontainingthestandardendotoxinataconcentrationof2,respectively.jC：2组装精细菌内毒素检测用水的4个试管，每个试管分别含有2人、入、入、入的内毒素标准品。dSolutionD:WaterforBET(negativeContrOI)溶液D：细菌内毒素检测用水（阴性对照）CalculationandInterpretation-Thetestisconsideredv
36、alidwhenthefollowingthreeconditionsaremet:(1)BothreplicatesofnegativecontrolSolutionDarenegative;(2)BothreplicatesofpositiveproductcontrolSolutionBarepositive;and(3)ThegeometricmeanendpointconcentrationofSolution。入to2人.计算和说明：只有满足以下三个条件时，化验才有效：阴性对照落彼。的两次检测结果均为阴性;(2)阳性产品对照溶液8的两次检测结果均为阳性；(3)溶液C的几何平均反响终
37、点浓度在入至2人的范围内。TodeterminetheendotoxinconcentrationofSolutionA,calculatetheendpointconcentrationforeachreplicatebymultiplyingeachendpointdilutionfactorby.TheendotoxinconcentrationintheSampleSolutionistheendpointconcentrationofthereplicates.IfthetestisconductedwithadilutedSampleSolution,calculatethecon
38、centrationofendotoxinintheoriginalSampleSolutionbymultiplyingbythedilutionfactor.IfnoneofthedilutionsoftheSampleSolutionispositiveinavalidassay,reporttheendotoxinconcentrationaslessthan(ifthedilutedsamplewastested,reportaslessthantimesthelowestdilutionfactorofthesample.)Ifalldilutionsarepositive,the
39、endotoxinconcentrationisreportedasequaltoorgreaterthanthegreatestdilutionfactormultipliedby(e.g.,initialdilutionfactortimes8times入inTabIe3).为了测定溶液4中的内毒素浓度,以每一个反响终点的稀释因子乘以入来计算每个重复测试的反响终点浓度。般落滋中的内毒素浓度是这些重复测试的反响终点浓度。如果是用稀释后的殍麻溶液进行的该试验，要计算初始楮7容滋中的内毒素浓度，就要乘以其稀释因子。如果在一个有效化验中，所有楮7容滋的稀释液均不呈阳性，那么就将内毒素浓度报告为少于入(如果是对稀释后的樗47容滋进行的检测，那么内毒素浓度检测结果那么应报告为少于样品最低稀释因子的1倍)。如果所有的稀释液均为阳性，将内毒素浓度报告为等于或大于最大稀释因子与人的乘积(例如，表3中，初始稀释因子乘以8再乘以)Thepreparationundertestmeetstherequirementsofthetestiftheconcentrationofendotoxininbothreplicatesislessthanthatspecifiedintheindividualmonograph.如果两次重复试验的内毒素浓度均小于专论中的限度值，那么这该溶液配制符合检测要求。
