小鼠尿激酶型纤溶酶原激活物受体试剂盒说明书

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2018-09-27 10:00 465阅读次数

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• 小鼠尿激酶型纤溶酶原激活物受体试剂盒说明书?

  小鼠尿激酶型纤溶酶原激活物受体试剂盒说明书?[详细]

  2016-01-20 00:00

  选购指南

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• 小鼠尿激酶型纤溶酶原激活物受体试剂盒说明书

  试验原理：uPA-R试剂盒是固相夹心法酶联免疫吸附实验（ELISA）.已知uPA-R浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。先将uPA-R和生物素标记的抗体同时温育。洗涤后，加入亲和素标记过的HRP。再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。产生颜色。颜色的深浅和样品中uPA-R的浓度呈比例关系。试剂盒内容及其配制试剂盒成份（2-8℃保存）96孔配置48孔配置配制96/48人份酶标板1块板（96T）半块板（48T）即用型塑料膜板盖1块半块即用型标准品：40ng/ml1瓶（0.6ml）1瓶（0.3ml）按说明书进行稀稀空白对照1瓶（1.0ml）1瓶（0.5ml）即用型标准品稀释缓冲液1瓶（4.0ml）1瓶（2.0ml）即用型生物素标记的抗uPA-R抗体1瓶（6.0ml）1瓶（3.0ml）即用型亲和链酶素-HRP1瓶（8.0ml）1瓶（4.0ml）即用型洗涤缓冲液1瓶（20ml）1瓶（10ml）按说明书进行稀释底物A1瓶（6.0ml）1瓶（3.0ml）即用型底物B1瓶（6.0ml）1瓶（3.0ml）即用型终止液1瓶（6.0ml）1瓶（3.0ml）即用型标本稀释液1瓶（12ml）1瓶（6.0ml）即用型自备材料1．蒸馏水。2．加样器：5ul、10ul、50ul、100ul、200ul、500ul、1000ul。3．振荡器及磁力搅拌器等。小鼠尿激酶型纤溶酶原激活物受体试剂盒安全性1．避免直接接触终止液和底物A、B。一旦接触到这些液体，请尽快用水冲洗。2．实验中不要吃喝、抽烟或使用化妆品。3．不要用嘴吸取试剂盒里的任何成份。操作注意事项1．试剂应按标签说明书储存，使用前恢复到室温。稀稀过后的标准品应丢弃，不可保存。2．实验中不用的板条应立即放回包装袋中，密封保存，以免变质。3．不用的其它试剂应包装好或盖好。不同批号的试剂不要混用。保质前使用。4．使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。5．使用干净的塑料容器配置洗涤液。使用前充分混匀试剂盒里的各种成份及样品。6．洗涤酶标板时应充分拍干，不要将吸水纸直接放入酶标反应孔中吸水。7．底物A应挥发，避免长时间打开盖子。底物B对光敏感，避免长时间暴露于光下。避免用手接触，有毒。实验完成后应立即读取OD值。8．加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。9．按照说明书中标明的时间、加液的量及顺序进行温育操作。样品收集、处理及保存方法1、血清-----操作过程中避免任何细胞刺激。使用不含热原和内毒素的试管。收集血液后，1000×g离心10分钟将血清和红细胞迅速小心地分离。2、血浆-----EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。3、细胞上清液---1000×g离心10分钟去除颗粒和聚合物。4、组织匀浆-----将组织加入适量生理盐水捣碎。1000×g离心10分钟，取上清液5、保存------如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒，检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。小鼠尿激酶型纤溶酶原激活物受体试剂盒试剂的准备1．标准品：标准品的系列稀释应在实验时准备，不能储存。稀释前将标准品振荡混匀。稀释比例按下表中进行：40ng/ml（6号标准品）原倍浓度不用稀释直接加入50ul。20ng/ml（5号标准品）100ul的原倍标准品加入100ul的标准品稀释液10ng/ml（4号标准品）100ul的5号标准品加入100ul的标准品稀释液5.0ng/ml（3号标准品）100ul的4号标准品加入100ul的标准品稀释液2.5ng/ml（2号标准品）100ul的3号标准品加入100ul的标准品稀释液1.25ng/ml（1号标准品）100ul的2号标准品加入100ul的标准品稀释液0ng/ml（空白对照）原始浓度不用稀释直接加入50ul。2．洗涤缓冲液（50×）的稀释：蒸馏水50倍稀释。操作步骤1．使用前，将所有试剂充分混匀。不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。2．根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。标本用标本稀释液1：1稀释后加入50ul于反应孔内。3．加入稀释好后的标准品50ul于反应孔、加入待测样品50ul于反应孔内。立即加入50ul的生物素标记的抗体。盖上膜板，轻轻振荡混匀，37℃温育1小时。4．甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作3次。如果用洗板机洗涤，洗涤次数增加一次。5．每孔加入60ul的亲和链酶素-HRP，轻轻振荡混匀，37℃温育30分钟。6．甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作3次。如果用洗板机洗涤，洗涤次数增加一次。7．每孔加入底物A、B各50ul，轻轻振荡混匀，37℃温育10分钟。避免光照。8．取出酶标板，迅速加入50ul终止液，加入终止液后应立即测定结果。9．在450nm波长处测定各孔的OD值。建议使用的实验方案标准品浓度（ng/ml）A4040样品样品样品样品样品样品样品样品样品样品B2020样品样品样品样品样品样品样品样品样品样品C1010样品样品样品样品样品样品样品样品样品样品D5.05.0样品样品样品样品样品样品样品样品样品样品E2.52.5样品样品样品样品样品样品样品样品样品样品F1.251.25样品样品样品样品样品样品样品样品样品样品G00样品样品样品样品样品样品样品样品样品样品H样品样品样品样品样品样品样品样品样品样品样品样品局限6号标准品以上的结果为非线性的，根据此标准曲线无法得到极ng确的结果。试剂盒性能1.灵敏度：Z小的检测浓度小于1号标准品。稀释度的线性。样品线性回归与预期浓度相关系数R值为0.990。2.特异性：不与其它细胞因子反应。3.重复性：板内、板间变异系数均小于10%。小鼠尿激酶型纤溶酶原激活物受体试剂盒结果判断与分析1、仪器值：于波长450nm的酶标仪上读取各孔的OD值2、以吸光度OD值为纵坐标（Y），相应的uPA-R标准品浓度为横坐标（X），做得相应的曲线，样品的uPA-R含量可根据其OD值由标准曲线换算出相应的浓度。3、检测值范围：0-40ng/ml4、敏感度：0.1ng/ml[详细]

  2018-09-27 10:00

  产品样册

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• 小鼠尿激酶型纤溶酶原激活物受体（uPA-R）ELISA试剂盒

  试验原理：uPA-R试剂盒是固相夹心法酶联免疫吸附实验（ELISA）.已知uPA-R浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。先将uPA-R和生物素标记的抗体同时温育。洗涤后，加入亲和素标记过的HRP。再经过温育和洗涤，去除未结合的酶结合物，然后加入底物A、B，和酶结合物同时作用。产生颜色。颜色的深浅和样品中uPA-R的浓度呈比例关系。试剂盒内容及其配制试剂盒成份（2-8℃保存）96孔配置48孔配置配制96/48人份酶标板1块板（96T）半块板（48T）即用型塑料膜板盖1块半块即用型标准品：40ng/ml1瓶（0.6ml）1瓶（0.3ml）按说明书进行稀稀空白对照1瓶（1.0ml）1瓶（0.5ml）即用型标准品稀释缓冲液1瓶（4.0ml）1瓶（2.0ml）即用型生物素标记的抗uPA-R抗体1瓶（6.0ml）1瓶（3.0ml）即用型亲和链酶素-HRP1瓶（8.0ml）1瓶（4.0ml）即用型洗涤缓冲液1瓶（20ml）1瓶（10ml）按说明书进行稀释底物A1瓶（6.0ml）1瓶（3.0ml）即用型底物B1瓶（6.0ml）1瓶（3.0ml）即用型终止液1瓶（6.0ml）1瓶（3.0ml）即用型标本稀释液1瓶（12ml）1瓶（6.0ml）即用型自备材料1．蒸馏水。2．加样器：5ul、10ul、50ul、100ul、200ul、500ul、1000ul。3．振荡器及磁力搅拌器等。小鼠尿激酶型纤溶酶原激活物受体(uPA-R)ELISA试剂盒安全性1．避免直接接触终止液和底物A、B。一旦接触到这些液体，请尽快用水冲洗。2．实验中不要吃喝、抽烟或使用化妆品。3．不要用嘴吸取试剂盒里的任何成份。操作注意事项1．试剂应按标签说明书储存，使用前恢复到室温。稀稀过后的标准品应丢弃，不可保存。2．实验中不用的板条应立即放回包装袋中，密封保存，以免变质。3．不用的其它试剂应包装好或盖好。不同批号的试剂不要混用。保质前使用。4．使用一次性的吸头以免交叉污染，吸取终止液和底物A、B液时，避免使用带金属部分的加样器。5．使用干净的塑料容器配置洗涤液。使用前充分混匀试剂盒里的各种成份及样品。6．洗涤酶标板时应充分拍干，不要将吸水纸直接放入酶标反应孔中吸水。7．底物A应挥发，避免长时间打开盖子。底物B对光敏感，避免长时间暴露于光下。避免用手接触，有毒。实验完成后应立即读取OD值。8．加入试剂的顺序应一致，以保证所有反应板孔温育的时间一样。9．按照说明书中标明的时间、加液的量及顺序进行温育操作。样品收集、处理及保存方法1、血清-----操作过程中避免任何细胞刺激。使用不含热原和内毒素的试管。收集血液后，1000×g离心10分钟将血清和红细胞迅速小心地分离。2、血浆-----EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。3、细胞上清液---1000×g离心10分钟去除颗粒和聚合物。4、组织匀浆-----将组织加入适量生理盐水捣碎。1000×g离心10分钟，取上清液5、保存------如果样品不立即使用，应将其分成小部分-70℃保存，避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒，检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。小鼠尿激酶型纤溶酶原激活物受体(uPA-R)ELISA试剂盒试剂的准备1．标准品：标准品的系列稀释应在实验时准备，不能储存。稀释前将标准品振荡混匀。稀释比例按下表中进行：40ng/ml（6号标准品）原倍浓度不用稀释直接加入50ul。20ng/ml（5号标准品）100ul的原倍标准品加入100ul的标准品稀释液10ng/ml（4号标准品）100ul的5号标准品加入100ul的标准品稀释液5.0ng/ml（3号标准品）100ul的4号标准品加入100ul的标准品稀释液2.5ng/ml（2号标准品）100ul的3号标准品加入100ul的标准品稀释液1.25ng/ml（1号标准品）100ul的2号标准品加入100ul的标准品稀释液0ng/ml（空白对照）原始浓度不用稀释直接加入50ul。2．洗涤缓冲液（50×）的稀释：蒸馏水50倍稀释。操作步骤1．使用前，将所有试剂充分混匀。不要使液体产生大量的泡沫，以免加样时加入大量的气泡，产生加样上的误差。2．根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空白孔建议做复孔。每个样品根据自己的数量来定，能使用复孔的尽量做复孔。标本用标本稀释液1：1稀释后加入50ul于反应孔内。3．加入稀释好后的标准品50ul于反应孔、加入待测样品50ul于反应孔内。立即加入50ul的生物素标记的抗体。盖上膜板，轻轻振荡混匀，37℃温育1小时。4．甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作3次。如果用洗板机洗涤，洗涤次数增加一次。5．每孔加入60ul的亲和链酶素-HRP，轻轻振荡混匀，37℃温育30分钟。6．甩去孔内液体，每孔加满洗涤液，振荡30秒，甩去洗涤液，用吸水纸拍干。重复此操作3次。如果用洗板机洗涤，洗涤次数增加一次。7．每孔加入底物A、B各50ul，轻轻振荡混匀，37℃温育10分钟。避免光照。8．取出酶标板，迅速加入50ul终止液，加入终止液后应立即测定结果。9．在450nm波长处测定各孔的OD值。建议使用的实验方案标准品浓度（ng/ml）A4040样品样品样品样品样品样品样品样品样品样品B2020样品样品样品样品样品样品样品样品样品样品C1010样品样品样品样品样品样品样品样品样品样品D5.05.0样品样品样品样品样品样品样品样品样品样品E2.52.5样品样品样品样品样品样品样品样品样品样品F1.251.25样品样品样品样品样品样品样品样品样品样品G00样品样品样品样品样品样品样品样品样品样品H样品样品样品样品样品样品样品样品样品样品样品样品局限6号标准品以上的结果为非线性的，根据此标准曲线无法得到极ng确的结果。试剂盒性能1.灵敏度：Z小的检测浓度小于1号标准品。稀释度的线性。样品线性回归与预期浓度相关系数R值为0.990。2.特异性：不与其它细胞因子反应。3.重复性：板内、板间变异系数均小于10%。结果判断与分析1、仪器值：于波长450nm的酶标仪上读取各孔的OD值2、以吸光度OD值为纵坐标（Y），相应的uPA-R标准品浓度为横坐标（X），做得相应的曲线，样品的uPA-R含量可根据其OD值由标准曲线换算出相应的浓度。3、检测值范围：0-40ng/ml4、敏感度：0.1ng/ml[详细]

  2018-09-27 10:00

  产品样册

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• 人可溶性尿激酶型纤溶酶原激活物受体试剂盒使用方法

  人可溶性尿激酶型纤溶酶原激活物受体试剂盒使用方法[详细]

  2016-09-06 00:00

  产品样册

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• 人可溶性尿激酶型纤溶酶原激活物受体 试剂盒使用方法

  本试剂盒仅供研究使用。检测范围：96T[详细]

  2018-10-01 10:00

  产品样册

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• 小鼠尿激酶型纤溶酶原激活物受体(PLAUR/uPAR)ELISA试剂盒elisa试剂盒说明书

  小鼠尿激酶型纤溶酶原激活物受体(PLAUR/uPAR)ELISA试剂盒elisa试剂盒说明书[详细]

  2024-10-08 05:27

  期刊论文

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• 人尿激酶型纤溶酶原激活物受体（PLAUR/uPAR）Elisa试剂盒说明书

  FORINVITROUSEANDRESEARCHUSEONLYNOTFORUSEINDIAGNOSTICORTHERAPEUTICPROCEDURES7thEdition（RevisedinNovember,2011）[INTENDEDUSE]ThekitisasandwichenzymeimmunoassayforinvitroquantitativemeasurementofuPARinhumanserum,plasmaandotherbiologicalfluids.[REAGENTSANDMATERIALSPROVIDED]ReagentsQuantityReagentsQuantityPre-coated,readytouse96-wellstripplate1Platesealerfor96wells4Standard（lyophilized）2StandardDiluent1×20mLDetectionReagentA（green）1×120μLAssayDiluentA（2×concentrate）1×6mLDetectionReagentB（red）1×120μLAssayDiluentB（2×concentrate）1×6mLTMBSubstrate1×9mLStopSolution1×6mLWashBuffer（30×concentrate）1×20mLInstructionmanual1[MATERIALSREQUIREDBUTNOTSUPPLIED]1.Microplatereaderwith450±10nmfilter.2.Precisionsingleormulti-channelpipettesanddisposabletips.3.EppendorfTubesfordilutingsamples.4.Deionizedordistilledwater.5.Absorbentpaperforblottingthemicrotiterplate.6.ContainerforWashSolution[STORAGEOFTHEKITS]1.Forunopenedkit:Allthereagents首ldbekeptaccordingtothelabelsonvials.TheStandard,DetectionReagentA,DetectionReagentBandthe96-wellstripplate首ldbestoredat-20oCuponreceiptwhiletheothers首ldbeat4oC.2.Foropenedkit:Whenthekitisopened,theremainingreagentsstillneedtobestoredaccordingtotheabovestoragecondition.Besides,pleasereturntheunusedwellstothefoilpouchcontainingthedesiccantpack,andresealalongentireedgeofzip-seal.Note:Itishighlyrecommendedtousetheremainingreagentswithin1monthprovidedthisiswithintheexpirationdateofthekit.[SAMPLECOLLECTIONANDSTORAGE]Serum-Allowsamplestoclotfortwohoursatroomtemperatureorovernightat4oCbeforecentrifugationfor20minutesatapproximately1000×g.Assayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gwithin30minutesofcollection.Removeplasmaandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Otherbiologicalfluids-Centrifugesamplesfor20minutesat1000×g.Removeparticulatesandassayimmediatelyorstoresamplesinaliquotat-20oCor-80oC.Avoidrepeatedfreeze/thawcycles.Note:1.Samplestobeusedwithin5daysmaybestoredat4oC,otherwisesamplesmustbestoredat-20oC（1month）or-80oC（2months）toavoidlossofbioactivityandcontamination.2.Samplehemolysiswillinfluencetheresult,sohemolyticspecimencannotbedetected.3.Whenperformingtheassay,bringsamplestoroomtemperature.[REAGENTPREPARATION]1.Bringallkitcomponentsandsamplestoroomtemperature（18-25oC）beforeuse.2.Standard-ReconstitutetheStandardwith1.0mLofStandardDiluent,keptfor10minutesatroomtemperature,shakegently（nottofoam）.Theconcentrationofthestandardinthestocksolutionis200ng/mL.Pleasefirstlydilutethestocksolutionto20ng/mLandthedilutedstandardservesasthehigheststandard（20ng/mL）.Thenprepare7tubescontaining0.5mLStandardDiluentandusethedilutedstandardtoproduceadoubledilutionseriesaccordingtothepictureshownbelow.Mixeachtubethoroughlybeforethenexttransfer.Setup7pointsofdilutedstandardsuchas20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL,andthelastEPtubeswithStandardDiluentistheblankas0ng/mL.3.AssayDiluentAandAssayDiluentB-Dilute6mLofAssayDiluentAorBConcentrate（2×）with6mLofdeionizedordistilledwatertoprepare12mLofAssayDiluentAorB.（Infact,morethan6mLAssayDiluentAandAssayDiluentBarecontainedinthebottles.Therefore,ineverytest,pleasepreciselypipetterequiredamountofDiluentandmakedoubledilutioninanewcontainer.Thepreparedworkingdilutioncan'tbefrozen.）Tube123456789ng/mL200201052.51.250.6250.31204.DetectionReagentAandDetectionReagentB-BrieflyspinorcentrifugethestockDetectionAandDetectionBbeforeuse.DilutetotheworkingconcentrationwithworkingAssayDiluentAorB,respectively（1:100）.5.WashSolution-Dilute20mLofWashSolutionconcentrate（30×）with580mLofdeionizedordistilledwatertoprepare600mLofWashSolution（1×）.6.TMBsubstrate-Aspiratetheneededdosageofthesolutionwithsterilizedtipsanddonotdumptheresidualsolutionintothevialagain.Note:1.Makingserialdilutioninthewellsdirectlyisnotpermitted.2.Preparestandardwithin15minutesbeforeassay.Pleasedonotdissolvethereagentsat37oCdirectly.3.PleasecarefullyreconstituteStandardsorworkingDetectionReagentAandBaccordingtotheinstruction,andavoidfoamingandmixgentlyuntilthecrystalsarecompletelydissolved.Tominimizeimprecisioncausedbypipetting,usesmallvolumesandensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10μLforoncepipetting.4.ThereconstitutedStandards,DetectionReagentAandDetectionReagentBcanbeusedonlyonce.5.IfcrystalshaveformedintheWashSolutionconcentrate（30×）,warmtoroomtemperatureandmixgentlyuntilthecrystalsarecompletelydissolved.6.Contaminatedwaterorcontainerforreagentpreparationwillinfluencethedetectionresult.[SAMPLEPREPARATION]1.Uscn,Inc.isonlyresponsibleforthekititself,butnotforthesamplesconsumedduringtheassay.Theuser首ldcalculatethepossibleamountofthesamplesusedinthewholetest.Pleasereservesufficientsamplesinadvance.2.Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.3.Ifthesamplesarenotindicatedinthemanual,apreliminaryexperimenttodeterminethevalidityofthekitisnecessary.4.TissueorcellextractionsamplespreparedbychemicallysisbuffermaycauseunexpectedELISAresultsduetotheimpactsfromcertainchemicals.5.Duetothepossibilityofmismatchingbetweenantigenfromotheroriginandantibodyusedinourkits（e.g.,antibodytargetsconformationalepitoperatherthanlinearepitope）,somenativeorrecombinantproteinsfromothermanufacturersmaynotberecognizedbyourproducts.6.Influencedbythefactorsincludingcellviability,cellnumberorsamp领time,samplesfromcellculturesupernatantmaynotbedetectedbythekit.7.Freshsampleswithoutlongtimestorageisrecommendedforthetest.Otherwise,proteindegradationanddenaturalizationmayoccurinthosesamplesandfinallyleadtowrongresults.[ASSAYPROCEDURE]1.Determinewellsfordilutedstandard,blankandsample.Prepare7wellsforstandard,1wellforblank.Add100μLeachofdilutionsofstandard（readReagentPreparation）,blankandsamplesintotheappropriatewells.CoverwiththePlatesealer.Incubatefor2hoursat37oC.2.Removetheliquidofeachwell,don’twash.3.Add100μLofDetectionReagentAworkingsolutiontoeachwell.Incubatefor1hourat37oCaftercoveringitwiththePlatesealer.4.Aspiratethesolutionandwashwith350μLof1×WashSolutiontoeachwellusingasquirtbottle,multi-channelpipette,manifolddispenserorautowasher,andletitsitfor1~2minutes.Removetheremainingliquidfromallwellscompletelybysnappingtheplateontoabsorbentpaper.Totallywash3times.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstabsorbentpaper.5.Add100μLofDetectionReagentBworkingsolutiontoeachwell.Incubatefor30minutesat37oCaftercoveringitwiththePlatesealer.6.Repeattheaspiration/washprocessfortotal5timesasconductedinstep4.7.Add90μLofSubstrateSolutiontoeachwell.CoverwithanewPlatesealer.Incubatefor15-25minutesat37oC（Don'texceed30minutes）.Protectfromlight.TheliquidwillturnbluebytheadditionofSubstrateSolution.8.Add50μLofStopSolutiontoeachwell.TheliquidwillturnyellowbytheadditionofStopsolution.Mixtheliquidbytappingthesideoftheplate.Ifcolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.9.Removeanydropofwaterandfingerprintonthebottomoftheplateandconfirmthereisnobubbleonthesurfaceoftheliquid.Then,runthemicroplatereaderandconductmeasurementat450nmimmediately.Note:1.Assaypreparation:Keepappropriatenumbersofwellsfor1experimentandremoveextrawellsfrommicroplate.Restwells首ldberesealedandstoredat-20oC.2.SamplesorreagentsadditionPleaseusethefreshlypreparedStandard.Pleasecarefullyaddsamplestowellsandmixgentlytoavoidfoaming.Donottouchthewellwall.Foreachstepintheprocedure,totaldispensingtimeforadditionofreagentsorsamplestotheassayplate首ldnotexceed10minutes.Thiswillensureequalelapsedtimeforeachpipettingstep,withoutinterruption.Duplicationofallstandardsandspecimens,althoughnotrequired,isrecommended.Toavoidcross-contamination,changepipettetipsbetweenadditionsofstandards,samples,andreagents.Also,useseparatedreservoirsforeachreagent.3.Incubation:Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.Donotallowwellstosituncoveredforextendedperiodsbetweenincubationsteps.Oncereagentsareaddedtothewellstrips,DONOTletthestripsDRYatanytimeduringtheassay.Incubationtimeandtemperaturemustbecontrolled.4.Washing:Thewashprocedureiscritical.Completeremovalofliquidateachstepisessentialforgoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecantingandremoveanydropofwaterandfingerprintonthebottomoftheplate.Insufficientwashingwillresultinpoorprecisionandfalseelevatedabsorbancereading.5.Control领ofreactiontime:ObservethechangeofcolorafteraddingTMBSubstrate（e.g.observationonceevery10minutes）,ifthecoloristoodeep,addStopSolutioninadvancetoavoidexcessivelystrongreactionwhichwillresultininaccurateabsorbancereading.6.TMBSubstrateiseasilycontaminated.Pleaseprotectitfromlight.7.Theenvironmenthumiditywhichislessthan60%mighthavesomeeffectsonthefinalperformance,therefore,ahumidifierisrecommendedtobeusedatthatcondition.[TESTPRINCIPLE]Themicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictouPAR.Standardsorsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedantibodypreparationspecificforuPAR.Next,AvidinconjugatedtoHorseradishPeroxidase（HRP）isaddedtoeachmicroplatewellandincubated.AfterTMBsubstratesolutionisadded,onlythosewellsthatcontainuPAR,biotin-conjugatedantibodyandenzyme-conjugatedAvidinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofsulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm±10nm.TheconcentrationofuPARinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.[CALCULATIONOFRESULTS]Averagetheduplicatereadingsforeachstandard,control,andsamplesandsubtracttheaveragezerostandardopticaldensity.Createastandardcurveonlog-loggraphpaper,withuPARconcentrationonthey-axisandabsorbanceonthex-axis.Drawthebestfitstraightlinethroughthestandardpointsanditcanbedeterminedbyregressionanalysis.Usingsomeplotsoftware,forinstance,curveexpert1.30,isalsorecommended.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.[TYPICALDATA]Inordertomakethecalculationeasier,weplottheO.D.valueofthestandard（X-axis）againsttheknownconcentrationofthestandard（Y-axis）,althoughconcentrationistheindependentvariableandO.D.valueisthedependentvariable.However,theO.D.valuesofthestandardcurvemayvaryaccordingtotheconditionsofassayperformance（e.g.operator,pipettingtechnique,washingtechniqueortemperatureeffects）,plottinglogofthedatatoestablishstandardcurveforeachtestisrecommended.Typicalstandardcurvebelowisprovidedforreferenceonly.TypicalStandardCurveforHumanuPARELISA.[DETECTIONRANGE]0.312-20ng/mL.ThestandardcurveconcentrationsusedfortheELISA’swere20ng/mL,10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL.[SENSITIVITY]TheminimumdetectabledoseofhumanuPARistypicallylessthan0.123ng/mL.Thesensitivityofthisassay,orLowerLimitofDetection（LLD）wasdefinedasthelowestproteinconcentrationthatcouldbedifferentiatedfromzero.Itwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.[SPECIFICITY]ThisassayhashighsensitivityandexcellentspecificityfordetectionofhumanuPAR.Nosignificantcross-reactivityorinterferencebetweenhumanuPARandanalogueswasobserved.Note:Limitedbycurrentskillsandknowledge,itisimpossibleforustocompletethecross-reactivitydetectionbetweenhumanuPARandalltheanalogues,therefore,crossreactionmaystillexist.[RECOVERY]MatriceslistedbelowwerespikedwithcertainlevelofrecombinanthumanuPARandtherecoveryrateswerecalculatedbycomparingthemeasuredvaluetotheexpectedamountofuPARinsamples.MatrixRecoveryrange（%）Average（%）humanserum（n=5）91-10597humanEDTAplasma（n=5）95-10498humanheparinplasma（n=5）80-9688[LINEARITY]ThelinearityofthekitwasassayedbytestingsamplesspikedwithappropriateconcentrationofhumanuPARandtheirserialdilutions.Theresultsweredemonstratedbythepercentageofcalculatedconcentrationtotheexpected.Sample121418116humanserum（n=5）95-109%80-91%91-107%81-95%humanEDTAplasma（n=5）93-101%76-99%90-99%83-93%humanheparinplasma（n=5）78-96%94-107%101-106%89-98%[PRECISION]Intra-assayPrecision（Precisionwithinanassay）:3sampleswithlow,middleandhighlevelhumanuPARweretested20timesononeplate,respectively.Inter-assayPrecision（Precisionbetweenassays）:3sampleswithlow,middleandhighlevelhumanuPARweretestedon3differentplates,8replicatesineachplate.CV（%）=SD/meanX100Intra-Assay:CV<10%Inter-Assay:CV<12%[STABILITY]ThestabilityofELISAkitisdeterminedbythelossrateofactivity.Thelossrateofthiskitislessthan5%withintheexpirationdateunderappropriatestoragecondition.Thelossratewasdeterminedbyacceleratedthermaldegradationtest.Keepthekitat37oCfor3days,andcompareO.D.valuesofthekitkeptat37oCwiththatofatrecommendedtemperature.（referringfromChinaBiologicalProductsStandard,whichwascalculatedbytheArrheniusequation.ForELISAkit,1daystorageat37oCcanbeconsideredas2monthsat4oC,whichmeans3daysat37oCequa领6monthsat4oC）.Note:Tominimizeextrainfluenceontheperformance,operationproceduresandlabconditions,especiallyroomtemperature,airhumidity,incubatortemperature首ldbestrictlycontrolled.Itisalsostronglysuggestedthatthewholeassayisperformedbythesameoperatorfromthebeginningtotheend.[ASSAYPROCEDURESUMMARY]1.Prepareallreagents,samplesandstandards;2.Add100μLstandardorsampletoeachwell.Incubate2hoursat37oC;3.Add100μLpreparedDetectionReagentA.Incubate1hourat37oC;4.Aspirateandwash3times;5.Add100μLpreparedDetectionReagentB.Incubate30minutesat37oC;6.Aspirateandwash5times;7.Add90μLSubstrateSolution.Incubate15-25minutesat37oC;8.Add50μLStopSolution.Readat450nmimmediately.[IMPORTANTNOTE]1.Limitedbythecurrentconditionandscientifictechnology,wecan'tcompletelyconductthecomprehensiveidentificationandanalysisontherawmaterialprovidedbysuppliers.Sotheremightbesomequalitativeandtechnicalriskstousethekit.2.Thefinalexperimentalresultswillbecloselyrelatedtovalidityoftheproducts,operationskillsoftheendusersandtheexperimentalenvironments.Pleasemakesurethatsufficientsamplesareavailable.3.Kitsfromdifferentbatchesmaybealittledifferentindetectionrange,sensitivityandcolordevelopingtime.Pleaseperformtheexperimentexactlyaccordingtotheinstructionattachedinkitwhileelectroniconesfromourwebsite（www.uscnk.us;www.uscnk.cn;www.uscnk.com）isonlyforinformation.4.Donotmixorsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.5.Protectallreagentsfromstronglightduringstorageandincubation.Allthebottlecapsofreagents首ldbecoveredtightlytopreventtheevaporationandcontaminationofmicroorganism.6.Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnothaveanyeffectonthefinalassayresults.Donotremovemicrotiterplatefromthestoragebaguntilneeded.7.Wrongoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingfortheplatereadermayleadtoincorrectresults.Amicroplateplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3O.D.orgreaterat450±10nmwavelengthisacceptableforuseinabsorbancemeasurement.Pleasereadtheinstructioncarefullyandadjusttheinstrumentpriortotheexperiment.Formoreinformation,pleaserefertotheoperationvideo（http://www.uscnk.com/homepage/operate-elisa.htm）.8.Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.Inordertogetbetterreproducibleresults,theoperationofeverystepintheassay首ldbecontrolled.Furthermore,apreliminaryexperimentbeforeassayforeachbatchisrecommended.9.EachkithasbeenstrictlypassedQ.Ctest.However,resultsfromendusersmightbeinconsistentwithourin-housedataduetosomeunexpectedtransportationconditionsordifferentlabequipments.Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromabovefactors,too.10.Kitsfromdifferentmanufacturerswiththesameitemmightproducedifferentresults,sincewehaven’tcomparedourproductswithothermanufacturers.11.Validperiod:sixmonths.12.Theinstructionmanualalsosuitsforthekitof48T,butallreagentsof48Tkitarereducedbyhalf.[PRECAUTION]TheStopSolutionsuggestedforusewiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.[详细]

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• （原装进口）人尿激酶型纤溶酶原激活物受体（uPAR）Elisa试剂盒说明书

  QuantikineHumanuPARImmunoassayCatalogNumberDUP00Forthequantitativedeterminationofhumanurokinase-typeplasminogenactivatorreceptor（uPAR）concentrationsincellculturesupernates,serum,plasma,andurine.Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.TABLEOFCONTENTSContentsPageINTRODUCTION2PRINCIPLEOFTHEASSAY..................................3LIMITATIONSOFTHEPROCEDURE3MATERIALSPROVIDED....................................3STORAGE4OTHERSUPPLIESREQUIRED.................................4PRECAUTION4SAMPLECOLLECTIONANDSTORAGE............................5SAMPLEPREPARATION5REAGENTPREPARATION...................................6ASSAYPROCEDURE7ASSAYPROCEDURESUMMARY...............................8CALCULATIONOFRESULTS9TYPICALDATA.........................................9TECHNICALHINTS10PRECISION...........................................10LINEARITY11SENSITIVITY..........................................11CALIBRATION11SAMPLEVALUES.......................................12SPECIFICITY13REFERENCES.........................................14PLATELAYOUT15MANUFACTUREDANDDISTRIBUTEDBY:R&DSystems,Inc.TELEPHONE:（800）343-7475614McKinleyPlaceNE（612）379-2956Minneapolis,MN55413FAX:（612）656-4400UnitedStatesofAmericaE-MAIL:info@RnDSystems.comDISTRIBUTEDBY:R&DSystemsEurope,Ltd.19BartonLaneTELEPHONE:+44（0）1235529449AbingdonScienceParkFAX:+44（0）1235533420Abingdon,OX143NBE-MAIL:info@RnDSystems.co.ukUnitedKingdomR&DSystemsChinaCo.Ltd.24A1HuaMinEmpirePlazaTELEPHONE:+86（21）52380373726WestYanAnRoadFAX:+86（21）52371001ShanghaiPRC200050E-MAIL:info@RnDSystemsChina.com.cnINTRODUCTIONUrokinase-typeplasminogenactivatorreceptor（uPAR,CD87）isacellsurfacereceptorthatbindsurokinase-typeplasminogenactivator（uPA）withhighaffinity,therebyfacilitatingthepericellularactivationofplasminogen（seereferences1and2forreviews）.uPAR,uPAandplasminogenactivatorinhibitor-1（PAI-1）,formatriadwithmultiplefunctions,includingregulationofcellattachment,migration,proliferationanddifferentiation,bybothproteolyticandnonproteolyticmechanisms（2）.uPARisanchoredtoextracellularsurfacesthroughaglycosylphosphatidylinositol（GPI）linkage,withnotransmembranedomain（3）.uPARissynthesizedasa335-amino-acidpolypeptidethatincludesa22-residuesignalpeptide（4）.A30-residuepeptideiscleavedfromtheC-terminusduringadditionoftheGPIanchor（3）.WithlossofthesignalpeptideandtheC-terminalpeptide,thematureproteinhas283aminoacids.Itisvariablyglycosylated,increasingitsmassfromabout31kDafortheproteinbackbonetoasmuchas55kDaforthematureglycoprotein（5）.Pro-uPA,asingle-chainprotein,isactivatedtoadisulfide-linked,two-chainproteinbyproteolyticcleavagebyplasmin（1,2,6）.Eitherpro-uPAortheactivetwo-chainuPAbindwithhighaffinitytouPAR.Thus,tracesofplasminactivatepro-uPAtouPA,leadingtoincreasinggenerationofplasmininapositivefeedbackloopthatisamplifiedbyuPAR.Whiletheinitiatingeventisnotclear,theeffectisthegenerationofplasminatthecellsurface,whereitdegradestheextracellularmatrixbyactivatingmatrixmetalloproteinases.Thisappearstobeakeyeventintumorinvasivenessandmetastasisandinmigrationofcellsingeneral（1,2,7）.ThefunctionsoftheuPA/uPARsystemare,however,moreextensivethanmediationofplasminformation,andtheyincludenon-proteolyticfunctions（1,2）.uPA/uPARislocalizedtofocalcontactpointsofcellswithinthesubstratum.Thesesitesincludeintracellularvinculinandtheextracellularadhesionmoleculevitronectin,suggestingdirectadhesivefunctionsandintracellularsignal领functionsforuPAR.uPARbindstointegrins,anditcontainschemotacticactivityinitssingleprotease-sensitiveregion.uPARhasbeenmeasuredinhumanplasma（7-9）.SolubleuPARisgeneratedbyremovaloftheGPIanchorbyanendogenousphospholipaseD,freeinguPARofitssurfaceattachment（10）.uPARiselevatedinplasmaofpatientswithparoxysmalnocturnalhemoglobinuria（7,8）,amanifestationofaninabilitytoaddGPIanchorstoproteins.IthasbeenpostulatedthattherealsomaybesolubleuPARduetoalternativesplicingoftheprimarytranscript（1）,ashasbeendemonstratedformouseuPAR（11）.uPARhasbeenidentifiedinurine,wherethelevelisaconsistentfractionofcreatinineconcentration（12）.TheQuantikineHumanuPARImmunoassayisa4.5hoursolid-phaseELISAdesignedtomeasurehumanuPARincellculturesupernates,serum,plasma,andurine.ItcontainsNS0-expressedrecombinanthumanuPARandantibodiesraisedagainsttherecombinantfactor.Ithasbeenshowntoaccuratelyquantitatetherecombinantfactor.ResultsobtainedusingnaturalhumanuPARshowedlinearcurvesthatwereparalleltothestandardcurvesobtainedusingtheQuantikinekitstandards.TheseresultsindicatethatthiskitcanbeusedtodeterminerelativemassvaluesofnaturalhumanuPAR.2PRINCIPLEOFTHEASSAYThisassayemploysthequantitativesandwichenzymeimmunoassaytechnique.AmonoclonalantibodyspecificforuPARhasbeenpre-coatedontoamicroplate.StandardsandsamplesarepipettedintothewellsandanyuPARpresentisboundbytheimmobilizedantibody.Afterwashingawayanyunboundsubstances,anenzyme-linkedpolyclonalantibodyspecificforuPARisaddedtothewells.Followingawashtoremoveanyunboundantibody-enzymereagent,asubstratesolutionisaddedtothewellsandcolordevelopsinproportiontotheamountofuPARboundintheinitialstep.Thecolordevelopmentisstoppedandtheintensityofthecolorismeasured.LIMITATIONSOFTHEPROCEDUREFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thekit首ldnotbeusedbeyondtheexpirationdateonthekitlabel.Donotmixorsubstitutereagentswithotherlotsorsources.Ifsamplesfalloutsidethedynamicrangeoftheassay,furtherdilutethesampleswithCalibratorDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.Thisassayisdesignedtoeliminateinterferencebyligandsandotherproteinspresentinbiologicalsamples.UntilallfactorshavebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.MATERIALSPROVIDEDuPARMicroplate（Part890714）-96wellpolystyrenemicroplate（12stripsof8wells）coatedwithamousemonoclonalantibodyagainstuPAR.uPARConjugate（Part890715）-21mLofpolyclonalantibodyagainstuPARconjugatedtohorseradishperoxidasewithpreservatives.uPARStandard（Part890716）-40ngofrecombinanthumanuPARinabufferedproteinbasewithpreservatives;lyophilized.AssayDiluentRD1W（Part895117）-11mLofabufferedproteinbasewithpreservatives.CalibratorDiluentRD6P（Part895118）-21mLofanimalserumwithpreservatives.WashBufferConcentrate（Part895003）-21mLofa25-foldconcentratedsolutionofbufferedsurfactantwithpreservatives.ColorReagentA（Part895000）-12.5mLofstabilizedhydrogenperoxide.ColorReagentB（Part895001）-12.5mLofstabilizedchromogen（tetramethylbenzidine）.StopSolution（Part895032）-6mLof2Nsulfuricacid.PlateCovers-4adhesivestrips.3STORAGEUnopenedKitStoreat2-8°C.Donotusepastkitexpirationdate.Opened/ReconstitutedReagentsDilutedWashBufferMaybestoredforupto1monthat2-8°C.*StopSolutionAssayDiluentRD1WCalibratorDiluentRD6PConjugateUnmixedColorReagentAUnmixedColorReagentBStandardMicroplateWellsReturnunusedwellstothefoilpouchcontainingthedesiccantpack,resealalongentireedgeofzip-seal.Maybestoredforupto1monthat2-8°C.**Providedthisiswithintheexpirationdateofthekit.OTHERSUPPLIESREQUIREDMicroplatereadercapableofmeasuringabsorbanceat450nm,withthecorrectionwavelengthsetat540nmor570nm.Pipettesandpipettetips.Deionizedordistilledwater.Squirtbottle,manifolddispenser,orautomatedmicroplatewasher.500mLgraduatedcylinder.Testtubesfordilution.HumanuPARControls（optional;availablefromR&DSystems）.PRECAUTIONTheStopSolutionprovidedwiththiskitisanacidsolution.Weareye,hand,face,andclothingprotectionwhenusingthismaterial.4SAMPLECOLLECTIONANDSTORAGECellCultureSupernates-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Serum-Useaserumseparatortube（SST）andallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesat1000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Plasma-CollectplasmausingheparinorEDTAasananticoagulant.Centrifugeat1000xgwithin30minutesofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.Avoidrepeatedfreeze-thawcycles.Note:Citrateplasmahasnotbeenvalidatedforuseinthisassay.LipemicsamplesandsamplescontaininghighlevelsofhumanserumalbuminarenotsuitableforthemeasurementofhumanuPARwiththisassay.Urine-Asepticallycollectthefirsturineoftheday（mid-stream）,voideddirectlyintoasterilecontainer.Centrifugetoremoveparticulatematter.Assayimmediatelyoraliquotandstoreat-20°C.Avoidrepeatedfreeze-thawcycles.Formoreinformationoncollectingandhand领urinespecimens,refertotheNationalCommitteeforClinicalLaboratoryStandards（NCCLS）publicationGP16-T.SAMPLEPREPARATIONCellculturesupernates,serum,plasma,andurinesamplesrequireatleasta5-folddilution.Asuggested5-folddilutionis25Lofsample+100LofCalibratorDiluentRD6P.5REAGENTPREPARATIONBringallreagentstoroomtemperaturebeforeuse.WashBuffer-Ifcrystalshaveformedintheconcentrate,warmtoroomtemperatureandmixgentlyuntilthecrystalshavecompletelydissolved.Dilute20mLofWashBufferConcentrateintodeionizedordistilledwatertoprepare500mLofWashBuffer.SubstrateSolution-ColorReagentsAandB首ldbemixedtogetherinequalvolumeswithin15minutesofuse.Protectfromlight.200Loftheresultantmixtureisrequiredperwell.uPARStandard-ReconstitutetheuPARStandardwith1.0mLofdeionizedordistilledwater.Thisreconstitutionproducesastocksolutionof40,000pg/mL.Allowthestandardtositforaminimumof15minuteswithgentleagitationpriortomakingdilutions.Pipette450LofCalibratorDiluentRD6Pintothe4000pg/mLtube.Pipette200LofCalibratorDiluentRD6Pintotheremainingtubes.Usethestocksolutiontoproduceadilutionseries（below）.Mixeachtubethoroughlybeforethenexttransfer.The4000pg/mLstandardservesasthehighstandard.CalibratorDiluentRD6Pservesasthezerostandard（0pg/mL）.6ASSAYPROCEDUREBringallreagentsandsamplestoroomtemperaturebeforeuse.Itisrecommendedthatallsamples,standards,andcontrolsbeassayedinduplicate.1.Prepareallreagents,workingstandards,andsamplesasdirectedintheprevioussections.2.Removeexcessmicroplatestripsfromtheplateframe,returnthemtothefoilpouchcontainingthedesiccantpack,andreseal.3.Add100LofAssayDiluentRD1Wtoeachwell.4.Add50LofStandard,control,orsample*perwell.Coverwiththeadhesivestripprovided.Incubatefor2hoursatroomtemperature.Aplatelayoutisprovidedtorecordstandardsandsamplesassayed.5.Aspirateeachwellandwash,repeatingtheprocessthreetimesforatotaloffourwashes.Washbyfil领eachwellwithWashBuffer（400L）usingasquirtbottle,manifolddispenser,orautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashBufferbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.Add200LofuPARConjugatetoeachwell.Coverwithanewadhesivestrip.Incubatefor2hoursatroomtemperature.7.Repeattheaspiration/washasinstep5.8.Add200LofSubstrateSolutiontoeachwell.Incubatefor30minutesatroomtemperature.Protectfromlight.9.Add50LofStopSolutiontoeachwell.Thecolorinthewells首ldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorifthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.10.Determinetheopticaldensityofeachwellwithin30minutes,usingamicroplatereadersetto450nm.Ifwavelengthcorrectionisavailable,setto540nmor570nm.Ifwavelengthcorrectionisnotavailable,subtractreadingsat540nmor570nmfromthereadingsat450nm.Thissubtractionwillcorrectforopticalimperfectionsintheplate.Readingsmadedirectlyat450nmwithoutcorrectionmaybehigherandlessaccurate.*Samplesrequiredilution.SeeSamplePreparationsection.7ASSAYPROCEDURESUMMARY8CALCULATIONOFRESULTSAveragetheduplicatereadingsforeachstandard,control,andsampleandsubtracttheaveragezerostandardopticaldensity.Createastandardcurvebyreducingthedatausingcomputersoftwarecapableofgeneratingalog/logcurve-fit.Asanalternative,constructastandardcurvebyplottingthemeanabsorbanceforeachstandardonthey-axisagainsttheconcentrationonthex-axisanddrawabestfitcurvethroughthepointsonalog/loggraph.ThedatamaybelinearizedbyplottingthelogoftheuPARconcentrationsversusthelogoftheO.D.onalinearscale,andthebestfitlinecanbedeterminedbyregressionanalysis.Ifthesampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.TYPICALDATAThisstandardcurveisprovidedfordemonstrationonly.Astandardcurve首ldbegeneratedforeachsetofsamplesassayed.9pg/mL062.5125250500100020004000O.D.0.0460.0440.0720.0730.1050.1050.1700.1710.3000.3040.5650.5451.0661.0752.1222.076Average0.0450.0720.1050.1700.3020.5551.0702.099Corrected___0.0270.0600.1250.2570.5101.0252.054TECHNICALHINTSWhenmixingorreconstitutingproteinsolutions,alwaysavoidfoaming.Toavoidcross-contamination,changepipettetipsbetweenadditionsofeachstandardlevel,betweensampleadditions,andbetweenreagentadditions.Also,useseparatereservoirsforeachreagent.Whenusinganautomatedplatewasher,addinga30secondsoakperiodfollowingtheadditionofwashbuffer,and/orrotatingtheplate180degreesbetweenwashstepsmayimproveassayprecision.Toensureaccurateresults,properadhesionofplatesealersduringincubationstepsisnecessary.SubstrateSolution首ldremaincolorlessuntiladdedtotheplate.KeeptheSubstrateSolutionprotectedfromlight.SubstrateSolution首ldchangefromcolorlesstogradationsofblue.StopSolution首ldbeaddedtotheplateinthesameorderastheSubstrateSolution.ThecolordevelopedinthewellswillturnfrombluetoyellowuponadditionoftheStopSolution.WellsthataregreenincolorindicatethattheStopSolutionhasnotmixedthoroughlywiththeSubstrateSolution.PRECISIONIntra-assayPrecision（Precisionwithinanassay）Threesamplesofknownconcentrationweretestedtwentytimesononeplatetoassessintra-assayprecision.Inter-assayPrecision（Precisionbetweenassays）Threesamplesofknownconcentrationweretestedinfortyseparateassaystoassessinter-assayprecision.Intra-assayPrecisionInter-assayPrecisionSample123123n202020404040Mean（pg/mL）8361593241279615462300Standarddeviation17.365.418144.378.9136CV（%）2.14.17.55.65.15.910LINEARITYToassessthelinearityoftheassay,samplesspikedwithhighconcentrationsofuPARweredilutedwithCalibratorDiluentRD6Ptoproducesampleswithvalueswithinthedynamicrangeoftheassay.Cellculturemedia*（n=4）Serum*（n=5）Heparinplasma*（n=5）EDTAplasma*（n=5）Urine*（n=5）1:2Average%ofExpected10396-112104101-105104101-10610194-105102Range（%）99-1051:4Average%ofExpected109101-115106103-111107104-110105101-108104Range（%）95-1121:8Average%ofExpected109101-111106103-112106102-110106103-113108Range（%）104-1131:16Average%ofExpected10999-11410599-11110598-11010498-111108Range（%）103-112*Sampleswerediluted5-foldpriortoassayasdirectedinSamplePreparation.SENSITIVITYTheminimumdetectabledose（MDD）ofuPARistypicallylessthan33pg/mL.TheMDDwasdeterminedbyaddingtwostandarddeviationstothemeanopticaldensityvalueoftwentyzerostandardreplicatesandcalculatingthecorrespondingconcentration.CALIBRATIONThisimmunoassayiscalibratedagainstahighlypurifiedNS0-expressedrecombinanthumanuPARproducedatR&DSystems.11SAMPLEVALUESSerum/Plasma/Urine-SamplesfromapparentlyhealthyvolunteerswereevaluatedforthepresenceofuPARinthisassay.Nomedicalhistorieswereavailableforthedonorsusedinthisstudy.SampleTypeMean（pg/mL）Range（pg/mL）Serum（n=60）23701195-4415Heparinplasma（n=35）2165977-5347EDTAplasma（n=35）1871864-3829Urine（n=35）2975691-6098CellCultureSupernates-Humanperipheralbloodmononuclearcells（5x106cells/mL）wereculturedinRPMIsupplementedwith5%fetalcalfserum,50M-mercaptoethanol,2mML-glutamine,100U/mLpenicillin,and100g/mLstreptomycinsulfate.Thecellswereculturedunstimulatedorstimulatedwith10g/mLPHA.AliquotsofthecellculturesupernateswereremovedandassayedforlevelsofnaturaluPAR.ConditionDay1（pg/mL）Day5（pg/mL）Unstimulated12282123Stimulated10,005689512SPECIFICITYThisassayrecognizesrecombinantandnaturalhumanuPAR.Thefactorslistedbelowwerepreparedat50ng/mLinCalibratorDiluentRD6Pandassayedforcross-reactivity.Preparationsofthefollowingfactorsat50ng/mLinamid-rangerecombinanthumanuPARcontrolwereassayedforinterference.Nosignificantcross-reactivityorinterferencewasobserved.RecombinanthumanuPAdoesnotcross-reactinthisassaybutdoesinterfereatconcentrationsgreaterthan3000pg/mL.13Recombinanthuman:ANGARCNTF-ECGFEGFEpoFGFacidicFGFbasicFGF-4FGF-5FGF-6Flt-3LigandG-CSFGM-CSFsgp130GROGROGROHB-EGFHGFIFN-IGF-IIGF-IIIL-1IL-1IL-1raIL-1sRIIL-1sRIIIL-2IL-2sRIL-3IL-3sRIL-4IL-4sRIL-5IL-5sRIL-5sRIL-6IL-6sRIL-7IL-8IL-9IL-10IL-11IL-12IL-13KGF（FGF-7）LAP（TGF-1）LIFM-CSFMCP-1MIP-1MIP-1-NGFOSMPD-ECGFPDGF-AAPDGF-ABPDGF-BBPTNRANTESSCFSLPITGF-TGF-1TGF-2TGF-3TGF-sRIITNF-TNF-sTNFRIsTNFRIIVEGFRecombinantmouse:GM-CSFIL-1IL-1IL-3IL-4IL-5IL-6IL-7IL-9IL-10IL-13LIFMIP-1MIP-1SCFTNF-uPARRecombinantamphibian:TGF-5Naturalproteins:bovineFGFacidicbovineFGFbasichumanPDGFporcinePDGFhumanTGF-1porcineTGF-1REFERENCES1.Dear,A.E.andR.L.Medcalf（1998）Eur.J.Biochem.252:185.2.Blasi,F.（1997）ImmunlogyToday18:415.3.Ploug,M.etal.（1991）J.Biol.Chem.266:1926.4.Roldan,A.L.（1990）EMBOJ.9:467.5.Behrendt,N.（1990）J.Biol.Chem.265:6453.6.Blasi,F.etal.（1987）J.CellBiol.104:801.7.Ronne,E.etal.（1995）Br.J.Haematol.89:576.8.Ploug,M.etal.（1992）Blood79:1447.9.Stephens,R.W.etal.（1999）J.Natl.CancerInst.91:869.10.Wilhelm,O.G.etal.（1999）J.CellularPhysiol.180:225.11.Kristensen,P.etal.（1991）J.CellBiol.115:1763.12.Sier,C.F.M.etal.（1999）Lab.Invest.79:717.14PLATELAYOUTUsethisplatelayouttorecordstandardsandsamplesassayed.2010R&DSystems,Inc.12.99750440.57/10[详细]

  2018-10-01 10:01

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• 大鼠尿激酶型纤溶酶原激活物(uPA)ELISA试剂盒

  大鼠尿激酶型纤溶酶原激活物(uPA)ELISA试剂盒[详细]

  2013-12-11 00:00

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• 人可溶性尿激酶型纤溶酶原激活物受体酶联免疫分析试剂盒使用方法

  本试剂盒仅供研究使用。检测范围：96T[详细]

  2018-12-30 10:00

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• 大鼠尿激酶型纤溶酶原激活物受体(PLAUR/uPAR)ELISA试剂

  大鼠尿激酶型纤溶酶原激活物受体(PLAUR/uPAR)ELISA试剂[详细]

  2024-09-17 19:14

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• 纤溶酶原激活物YZ物

  纤溶酶原激活物YZ物[详细]

  2024-09-28 01:25

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• 人纤溶酶原激活物YZ因子(PAI)ELISA试剂盒

  人纤溶酶原激活物YZ因子(PAI)ELISA试剂盒[详细]

  2013-12-06 00:00

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• 猪纤溶酶原激活物YZ因子(PAI)ELISA试剂盒

  猪纤溶酶原激活物YZ因子(PAI)ELISA试剂盒[详细]

  2024-09-17 11:02

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• 大鼠纤溶酶原激活物YZ因子(PAI)ELISA试剂盒

  大鼠纤溶酶原激活物YZ因子(PAI)ELISA试剂盒[详细]

  2024-09-29 00:38

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• 人纤溶酶原激活物YZ因子1(PAI-1)elisa试剂盒说明书

  人纤溶酶原激活物YZ因子1(PAI-1)elisa试剂盒说明书[详细]

  2024-10-08 05:28

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• 纤溶酶原激活物YZ因子1分析测试

  纤溶酶原激活物YZ因子1分析测试目的：本试剂盒用于测定大鼠血清，血浆及相关液体样本中纤溶酶原激活物YZ因子1(PAI-1)的含量。实验原理：本试剂盒应用双抗体夹心法测定标本中大鼠纤溶酶原激活物YZ因子1(PAI-1)水平。用纯化的大鼠纤溶酶原激活物YZ因子1(PAI-1)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入PAI-1，再与HRP标记的羊抗鼠抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成Z终的黄色。颜色的深浅和样品中的PAI-1呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中大鼠纤溶酶原激活物YZ因子1(PAI-1)浓度。纤溶酶原激活物YZ因子1分析测试纤溶酶原激活物YZ因子1分析测试大鼠组织多肽抗原(TPA)ELISA试剂盒人凝血酶受体(TR)ELISA试剂盒人巨噬细胞趋化因子(MCF)ELISA试剂盒小鼠心肌转录因子GATA4ELISA试剂盒大鼠低氧诱导因子1α(HIF-1α)ELISA试剂盒大鼠前列腺素E2(PGE2)ELISA试剂盒鸡血管内皮细胞生长因子(VEGF)ELISA试剂盒大鼠胰岛素样生长因子2(IGF-2)ELISA试剂盒人类乳头状瘤病毒抗体牛乙酰乙酸检测(ACAC)ELISA试剂盒鸡白介素4(IL-4)ELISA试剂盒克氏双糖铁琼脂（下层）250gBRV5tag标签抗体明胶25g培养基蛋白质原料单位：元货号产品名称规格价格备注BS7001胰蛋白胨250g32ARBS7002...人脊髓灰质炎病毒IgG(PV-IgG)ELISA试剂盒人神经趋化蛋白(fractalkine/CX3CL1)ELISA试剂盒人酰化刺激蛋白(ASP)ELISA试剂盒小鼠胰岛素自身抗体(IAA)ELISA试剂盒4-甲基伞形酮葡萄糖酸苷MUG培养基2000药典25gBR鱼雌激素（E）ELISA试剂盒大鼠可溶性肿瘤坏死因子相关凋亡诱导配体(sTRAIL)ELISA试剂盒猪载脂蛋白A1(apo-A1)ELISA试剂盒P53凋亡刺激蛋白2抗体纤溶酶原激活物YZ因子1分析测试[详细]

  2018-10-23 10:30

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• 人纤溶酶原激活物YZ因子1(PAI-1)ELISA试剂盒

  人纤溶酶原激活物YZ因子1(PAI-1)ELISA试剂盒[详细]

  2013-12-06 00:00

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• 人纤溶酶原激活物YZ剂1（PAI-1）ELISA试剂盒

  人纤溶酶原激活物YZ剂1(PAI-1)ELISA试剂盒操作步骤：1.标准品的稀释与加样：在酶标包被板上设标准品孔10孔，在**、第二孔中分别加标准品100μl，然后在**、第二孔中加标准品稀释液50μl，混匀；然后从**孔、第二孔中各取100μl分别加到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50μl，混匀；然后在第三孔和第四孔中先各取50μl弃掉，再各取50μl分别加到第五、第六孔中，再在第五、第六孔中分别加标准品稀释液50ul，混匀；混匀后从第五、第六孔中各取50μl分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50μl，混匀后从第七、第八孔中分别取50μl加到第九、第十孔中，再在第九第十孔分别加标准品稀释液50μl，混匀后从第九第十孔中各取50μl弃掉。（稀释后各孔加样量都为50μl，浓度分别为600ng/L，400ng/L，200ng/L，100ng/L，50ng/L）。2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品Z终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。3.温育：用封板膜封板后置37℃温育30分钟。4.配液：将30（48T的20倍）倍浓缩洗涤液用蒸馏水30（48T的20倍）倍稀释后备用。5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。6.加酶：每孔加入酶标试剂50μl，空白孔除外。7.温育：操作同3。8.洗涤：操作同5。9.显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色15分钟.10.终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。11.测定：以空白空调零，450nm波长依序测量各孔的吸光度（OD值）。测定应在加终止液后15分钟以内进行。[详细]

  2018-11-05 10:00

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• 大鼠纤溶酶原激活物YZ因子1(PAI-1)ELISA试剂盒

  大鼠纤溶酶原激活物YZ因子1(PAI-1)ELISA试剂盒[详细]

  2024-09-19 05:37

  课件

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