USP34-细菌内毒素《85》.docx

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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/SampleSolution无I样品溶液4BbikSampleSolution2入样品SampleSolution样品12人4溶液溶液21人