检测98成人网

2017年4月修订第七版Elabscience(本试剂盒仅供体外研究使用,不用于临床诊断!
)人血管紧张素Ⅱ受体1(ANGⅡR1)酶联免疫吸附测定试剂盒使用说明书HumanANGⅡR1(AngiotensinⅡReceptor1)ELISAKit产品货号:E-EL-H0329c96T使用前请仔细阅读说明书.
如果有任何问题,请通过以下方式联系我们:销售部电话027-65022280,027-87854967技术部电话027-87526315电子邮箱(销售)Perry@elabscience.
cn电子邮箱(技术)techsupport@elabscience.
cnQQ客服800110755网址:www.
elabscience.
cn具体保质期请见试剂盒外包装标签.
联系时请提供产品批号(见试剂盒标签),以便我们更高效地为您服务.
Copyright2017-2018ElabscienceBiotechnologyCo.
,Ltd.
AllRightsReserved2017年4月修订第七版用途该试剂盒用于体外定量检测人血清、血浆或其他相关生物液体中ANGⅡR1浓度.
灵敏度、检测范围、特异性和重复性灵敏度0.
10ng/mL检测范围:0.
16-10ng/mL特异性:可检测样本中的人ANGⅡR1,且与其类似物无明显交叉反应.
重复性:板内,板间变异系数均<10%.
检测原理本试剂盒采用双抗体夹心ELISA法.
用抗人ANGⅡR1抗体包被于酶标板上,实验时样品或标准品中的人ANGⅡR1会与包被抗体结合,游离成分被洗去.
依次加入生物素化的抗人ANGⅡR1抗体和辣根过氧化物酶标记亲和素.
抗人ANGⅡR1抗体与结合在包被抗体上的人ANGⅡR1结合、生物素与亲和素特异性结合而形成免疫复合物,游离的成分被洗去.
加入显色底物(TMB),TMB在辣根过氧化物酶的催化下呈现蓝色,加终止液后变成黄色.
用酶标仪在450nm波长处测OD值,ANGⅡR1浓度与OD450值之间呈正比,通过绘制标准曲线计算出样品中ANGⅡR1的浓度.
www.
elabscience.
cn22017年4月修订第七版试剂盒组成及保存未拆封的试剂盒可在2-8℃保存一周;如果一周以后才使用试剂盒,请拆开试剂盒按照下表中的条件分别保存各组分.
中文名称英文名称规格保存条件ELISA酶标板(可拆卸)MicroELISAPlate(Dismountable)8孔*12条-20℃,可存放6个月冻干标准品ReferenceStandard2支浓缩生物素化抗体(100*)ConcentratedBiotinylatedDetectionAb1支120μL浓缩HRP酶结合物(100*)ConcentratedHRPConjugate1支120μL-20℃(避光),可存放6个月标准品&样品稀释液ReferenceStandard&SampleDiluent1瓶20mL2-8℃,可存放6个月生物素化抗体稀释液BiotinylatedDetectionAbDiluent1瓶14mL酶结合物稀释液HRPConjugateDiluent1瓶14mL浓缩洗涤液(25*)ConcentratedWashBuffer(25*)1瓶30mL底物溶液(TMB)SubstrateReagent1瓶10mL2-8℃(避光)反应终止液StopSolution1瓶10mL2-8℃封板覆膜PlateSealer5张产品说明书ProductDescription1份质检报告CertificateofAnalysis1份说明:所有试剂瓶盖须旋紧以防止蒸发和微生物的污染.
试剂体积以实际发货版说明书为准.
相关试剂在分装时会比标签上标明的体积稍多一些,请在使用时量取而非直接倒出.
试验所需自备物品1.
酶标仪(450nm波长滤光片)2.
高精度移液器,EP管及一次性吸头:0.
5-10μL,2-20μL,20-200μL,200-1000μL3.
37℃恒温箱,双蒸水或去离子水4.
吸水纸注意事项1.
试验中请穿着实验服并戴乳胶手套做好防护工作.
特别是检测血液或者其他体液样品时,请按国家生物试验室安全防护条例执行.
2.
刚开启的酶标板孔中可能会有少许水样物质,此为正常现象,不会对实验结果造成任何影响.
暂时不用的板条应拆卸后放入备用铝箔袋,按照上述表格中保存条件存放.
3.
请勿重复使用已稀释过的标准品、生物素化抗体工作液、酶结合物工作液.
未用完的没有稀释的浓缩生物素化抗体(100*)、浓缩HRP酶结合物(100*)、酶标板及其他原液按照上述表格中保存条件存放.
4.
检测使用的酶标仪需要安装能检测450±10nm波长的滤光片,光密度范围在0-3.
5之间.
5.
不同批号的试剂盒组份不能混用(反应终止液除外).
6.
试验中所用的EP管和吸头均为一次性使用,严禁混用.
www.
elabscience.
cn32017年4月修订第七版样品收集方法(具体处理方法参考官网:http://www.
elabscience.
cn)1.
血清:全血样品于室温放置2小时或2-8℃过夜后于1000*g离心20分钟,取上清即可检测,收集血液的试管应为一次性的无内毒素试管.
2.
血浆:抗凝剂推荐使用EDTA钠盐,样品采集后30分钟内于1000*g离心15分钟,取上清即可检测.
避免使用溶血,高血脂样品.
3.
组织匀浆:用预冷的PBS(0.
01M,pH=7.
4)冲洗组织,去除残留血液,称重后将组织剪碎.
将剪碎的组织与对应体积的PBS(一般按1:9的重量体积比,比如1g的组织样品对应9mL的PBS,具体体积可根据实验需要适当调整,并做好记录.
推荐在PBS中加入蛋白酶抑制剂)加入玻璃匀浆器中,在冰上充分研磨.
为了进一步裂解组织细胞,可以对匀浆液进行超声破碎或反复冻融.
最后将匀浆液5000*g离心5-10分钟,取上清检测.
4.
细胞提取液:贴壁细胞用冷的PBS轻轻清洗,然后用胰蛋白酶消化,1000*g离心5分钟后收集细胞;悬浮细胞可直接离心收集.
收集的细胞用冷的PBS洗涤3次.
每1*106个细胞中加入150-200μLPBS重悬并通过反复冻融使细胞破碎(若含量很低可减少PBS的体积).
将提取液于1500*g离心10分钟,取上清检测.
5.
细胞培养上清或其他生物体液:1000*g离心20分钟,除去杂质及细胞碎片.
取上清检测.
样品注意事项1.
样品收集后若在1周内进行检测的可保存于2-8℃,若不能及时检测,请按一次使用量分装,冻存于-20℃(1个月内检测),或-80℃(3个月内检测),避免反复冻融.
2.
试剂盒检测范围不等同于样本的浓度范围,如果您的样品中检测物浓度高于标准品最高值,请根据实际情况,做适当倍数稀释(建议查阅文献后先做预实验,以确定稀释倍数).
3.
若所检样本不在说明书所列样本之中,建议做预实验验证其检测有效性.
4.
若使用化学裂解液制备组织匀浆或细胞提取液,由于引入某些化学物质会导致ELISA测值出现偏差.
5.
某些重组蛋白可能与试剂盒中捕获或检测抗体不匹配而出现不能检测的情况.
www.
elabscience.
cn42017年4月修订第七版检测前准备工作:1.
请提前20分钟从冰箱中取出试剂盒,平衡至室温.
读数前15分钟打开酶标仪预热.
2.
洗涤液:将浓缩洗涤液用双蒸水稀释(1:24).
提示:从冰箱中取出的浓缩洗涤液可能有结晶,属于正常现象,可用40℃水浴微加热使结晶完全溶解后再配制洗涤液.
当日使用.
3.
标准品工作液将标准品于10000*g离心1分钟,加入标准品&样品稀释液1.
0mL至冻干标准品中,旋紧管盖,静置10分钟,上下颠倒数次,待其充分溶解后,轻轻混匀,配成10ng/mL的标准品工作液.
然后根据需要进行倍比稀释.
建议配制成以下浓度:10,5,2.
5,1.
25,0.
63,0.
32,0.
16,0ng/mL.
倍比稀释方法:取7支EP管,每管中加入500μL标准品&样品稀释液,从10ng/mL的标准品工作液中吸取500μL到其中一支EP管中混匀配成5ng/mL的标准品工作液,按此步骤往后依次吸取混匀.
如下图.
提示:最后一管直接作为空白孔,不需要再从倒数第二管中吸取液体.
1052.
51.
250.
630.
320.
1604.
生物素化抗体工作液:实验前计算实验所需用量(以100μL/孔计算),实际配制时应多配制100-200μL.
使用前15分钟,以生物素化抗体稀释液将100*浓缩生物素化抗体稀释成1*工作浓度.
当日使用.
5.
酶结合物工作液:实验前计算当次实验所需用量(以100μL/孔计算),实际配制时应多配制100-200μL.
使用前15分钟,以酶结合物稀释液将100*浓缩HRP酶结合物稀释成1*工作浓度.
当日使用.
www.
elabscience.
cn52017年4月修订第七版操作步骤(第10页中附有简版操作概要)1.
将标准品工作液依次加入到前两列孔中,每个浓度的工作液并列加两孔,每孔100μL.
待测样品加入到其他孔,每孔100μL(若样本浓度高于检测范围,需用标准品&样本稀释液稀释后取样).
给酶标板覆膜,37℃孵育90分钟.
提示:加样时将样品加于酶标板底部,尽量不触及孔壁,轻轻晃动混匀,避免产生气泡.
加样时间控制在10分钟内.
2.
弃去液体,甩干,不用洗涤.
每个孔中加入生物素化抗体工作液100μL,混匀,酶标板加上覆膜,37℃温育1小时.
3.
甩尽孔内液体,每孔加洗涤液350μL,浸泡1-2分钟,吸去或甩掉酶标板内的液体,在厚的吸水纸上拍干.
重复此洗板步骤3次.
提示:此处与其他洗板步骤都可用洗板机.
4.
每孔加酶结合物工作液100μL,加上覆膜,37℃温育30分钟.
5.
弃去孔内液体,甩干,洗板5次,方法同步骤3.
6.
每孔加90μL底物溶液(TMB),酶标板加上覆膜37℃避光孵育15分钟左右.
提示:根据实际显色情况酌情缩短或延长,但不可超过30分钟.
当标准孔出现明显梯度时,即可终止.
7.
每孔加终止液50μL,终止反应.
提示:终止液的加入顺序应尽量与底物溶液的加入顺序相同.
8.
立即用酶标仪在450nm波长测量各孔的光密度(OD值).
结果判断1.
计算每组复孔的平均OD值.
每个标准品的平均OD值减去空白孔的OD值作为矫正值.
以浓度为横坐标,OD值为纵坐标,在双对数坐标纸上绘出四参数逻辑函数的标准曲线(作图时去掉空白组的值).
2.
若样品OD值高于标准曲线上限,应适当稀释后重测.
www.
elabscience.
cn62017年4月修订第七版典型数据由于实验操作条件的不同(如操作者、移液技术、洗板技术和稳定条件等),标准曲线的OD值会有所差异.
以下数据和曲线仅供参考,实验者需根据自己的实验建立标准曲线.
Concentration(ng/mL)1052.
51.
250.
630.
320.
160OD2.
4891.
7651.
0570.
5180.
2690.
1790.
1220.
065CorrectedOD2.
4241.
70.
9920.
4530.
2040.
1140.
057--www.
elabscience.
cn72017年4月修订第七版精密度板内精密度:低浓度样本,中浓度样本和高浓度样本分别在1块板子上检测20次.
板间精密度:低浓度样本,中浓度样本和高浓度样本分别在3块板子上检测20次.
Intra-assayPrecisionInter-assayPrecisionSample123123n202020202020Mean(ng/mL)0.
521.
034.
330.
531.
074.
76Standarddeviation0.
030.
060.
160.
030.
060.
15CV(%)5.
775.
833.
705.
665.
613.
15回收率分别往5个不同样本中添加已知浓度的目标蛋白,做回收实验,得出回收率范围和平均回收率.
SampleTypeRange(%)AverageRecovery(%)Serum(n=5)90-10798EDTAplasma(n=5)95-106100Cellculturemedia(n=5)96-111101线性分别往5个样本中添加已知浓度的目标蛋白,做回收实验,得出回收率范围及平均回收率.
将5个样本分别稀释2倍,4倍,8倍,16倍做回收实验,得出回收率范围及平均回收率.
Serum(n=5)EDTAplasma(n=5)Cellculturemedia(n=5)1:2Range(%)86-9888-10287-99Average(%)9294931:4Range(%)88-9986-9884-98Average(%)9493901:8Range(%)94-10685-9886-97Average(%)9991921:16Range(%)91-10383-9988-102Average(%)969095www.
elabscience.
cn82017年4月修订第七版问题分析若实验效果不好,请及时对显色结果拍照,保存实验数据,保留所用板条及未使用试剂,然后联系我公司技术支持为您解决问题.
同时您也可以参考以下资料:问题描述可能原因相应对策标准曲线梯度差吸液或加液不准检查移液器及吸头标准品稀释不正确溶解标准品时稍微旋转瓶身,轻轻混匀使粉末完全溶解洗涤不完全保证洗涤时间和洗涤次数及每孔的加液量显色很弱或无色孵育时间太短保证充足的孵育时间实验温度不正确使用推荐的实验温度试剂体积不够或漏加检查吸液及加液过程,保证所有试剂按顺序足量添加稀释不正确酶标记物失活或底物失效混合酶结合物和底物,通过迅速显色来检查判断读数数值低酶标仪设置不正确在酶标仪上检查波长及滤光片设置提前打开酶标仪预热变异系数大加液不正确检查加液情况背景值高检测抗体的工作浓度过高使用推荐的稀释倍数酶标板洗涤不完全保证每步清洗完全;如果用自动洗板机,请检查所有的出口是否有堵塞;是否使用试剂盒配备的洗涤液洗液有污染配制新鲜的洗液灵敏度低ELISA试剂盒保存不当按说明书要求保存相关试剂读数前未终止OD读数前应在每孔中加入终止液www.
elabscience.
cn92017年4月修订第七版操作概要1.
在各孔中加入标准品或样品各100μL,37℃孵育90分钟2.
倒去孔内液体,加入100μL生物素化抗体工作液,37℃孵育60分钟3.
洗涤3次4.
加入100μL酶结合物工作液,37℃孵育30分钟5.
洗涤5次6.
加入90μL底物溶液,37℃孵育15分钟左右7.
加入50μL终止液,立即在450nm波长处测量OD值8.
结果计算声明1.
限于现有条件及科学技术水平,尚不能对所有原料进行全面的鉴定分析,本产品可能存在一定的质量技术风险.
2.
最终的实验结果与试剂的有效性、实验者的相关操作以及当时的实验环境密切相关,请务必准备充足的待测样品.
www.
elabscience.
cn102017年4月修订第七版HumanANGⅡR1(AngiotensinⅡReceptor1)ELISAKitSynonyms:AT1R,AG2S,AG2-S,AGTR1A,AGTR1B,AT1,AT1B,AT2R1,AT2-R1,AT2R1A,AT2R1B,HAT1R,AngiotensinIIreceptor,type1,AT1receptor,CatalogNo:E-EL-H0329c96TIntendeduseThisELISAkitappliestotheinvitroquantitativedeterminationofHumanANGⅡR1concentrationsinserum,plasmaandotherbiologicalfluids.
SpecificationSensitivity:0.
10ng/mLDetectionRange:0.
16-10ng/mLSpecificity:ThiskitrecognizesHumanANGⅡR1insamples.
Nosignificantcross-reactivityorinterferencebetweenHumanANGⅡR1andanalogueswasobserved.
Repeatability:Coefficientofvariationis<10%.
TestprincipleThisELISAkitusestheSandwich-ELISAprinciple.
ThemicroELISAplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoHumanANGⅡR1.
StandardsorsamplesareaddedtothemicroELISAplatewellsandcombinedwiththespecificantibody.
ThenabiotinylateddetectionantibodyspecificforHumanANGⅡR1andAvidin-HorseradishPeroxidase(HRP)conjugateareaddedsuccessivelytoeachmicroplatewellandincubated.
Freecomponentsarewashedaway.
Thesubstratesolutionisaddedtoeachwell.
OnlythosewellsthatcontainHumanANGⅡR1,biotinylateddetectionantibodyandAvidin-HRPconjugatewillappearblueincolor.
Theenzyme-substratereactionisterminatedbytheadditionofstopsolutionandthecolorturnsyellow.
Theopticaldensity(OD)ismeasuredspectrophotometricallyatawavelengthof450nm±2nm.
TheODvalueisproportionaltotheconcentrationofHumanANGⅡR1.
YoucancalculatetheconcentrationofHumanANGⅡR1inthesamplesbycomparingtheODofthesamplestothestandardcurve.
www.
elabscience.
cn112017年4月修订第七版Kitcomponents&StorageAnunopenedkitcanbestoredat2-8℃for1week.
Ifthekitisnotusedwithin1week,storetheitemsseparatelyaccordingtothefollowingconditionsoncethekitisreceived.
ItemSpecificationsStorageMicroELISAPlate(Dismountable)8wells*12strips-20℃,6monthsReferenceStandard2vialsConcentratedBiotinylatedDetectionAb(100*)1vial,120μLConcentratedHRPConjugate(100*)1vial,120μL-20°C(shadinglight),6monthsReferenceStandard&SampleDiluent1vial,20mL4°C,6monthsBiotinylatedDetectionAbDiluent1vial,14mLHRPConjugateDiluent1vial,14mLConcentratedWashBuffer(25*)1vial,30mLSubstrateReagent1vial,10mL4°C(shadinglight)StopSolution1vial,10mL4°CPlateSealer5piecesProductDescription1copyCertificateofAnalysis1copyNote:Allreagentbottlecapsmustbetightenedtopreventevaporationandmicrobialpollution.
Thevolumeofreagentsinpartialshipmentsisalittlemorethanthevolumemarkedonthelabel,pleaseuseaccuratemeasuringequipmentinsteadofdirectlypouringintothevial(s).
OthersuppliesrequiredMicroplatereaderwith450nmwavelengthfilterHigh-precisiontransferpipette,EPtubesanddisposablepipettetipsIncubatorcapableofmaintaining37℃DeionizedordistilledwaterAbsorbentpaperLoadingslotforWashBufferwww.
elabscience.
cn122017年4月修订第七版Note1.
Pleasewearlabcoats,eyeprotectionandlatexglovesforprotection.
Pleaseperformtheexperimentfollowingthenationalsecurityprotocolsofbiologicallaboratories,especiallywhendetectingbloodsamplesorotherbodilyfluids.
2.
AfreshlyopenedELISAPlatemayappeartohaveawater-likesubstance,whichisnormalandwillnothaveanyimpactontheexperimentalresults.
3.
Donotreusethereconstitutedstandard,biotinylateddetectionAbworkingsolution,concentratedHRPconjugateworkingsolution.
TheunspentundilutedconcentratedbiotinylateddetectionAb(100*)andotherstocksolutionsshouldbestoredaccordingtothestorageconditionsintheabovetable.
4.
Themicroplatereadershouldhavea450(±10nm)filterinstalledandadetectorthatcandetectthewavelength.
Theopticaldensityshouldbewithin0~3.
5.
5.
Donotmixorusecomponentsfromotherlots.
6.
Changepipettetipsinbetweenaddingstandards,inbetweensampleadditions,andinbetweenreagentadditions.
Also,useseparatereservoirsforeachreagent.
SamplecollectionSerum:Allowsamplestoclotfor2hoursatroomtemperatureorovernightat2-8℃beforecentrifugationfor20minat1000*gat2-8℃.
Collectthesupernatanttocarryouttheassay.
Bloodcollectiontubesshouldbedisposableandbenon-endotoxin.
Plasma:CollectplasmausingEDTA-Na2asananticoagulant.
Centrifugesamplesfor15minat1000*gat2-8℃within30minofcollection.
Collectthesupernatanttocarryouttheassay.
HemolysedsamplesarenotsuitableforELISAassay!
Tissuehomogenates:Itisrecommendedtogetdetailedreferencesfromtheliteraturebeforeanalyzingdifferenttissuetypes.
Forgeneralinformation,hemolysedbloodmayaffecttheresults,sothetissuesshouldbemincedintosmallpiecesandrinsedinice-coldPBS(0.
01M,pH=7.
4)toremoveexcessbloodthoroughly.
TissuepiecesshouldbeweighedandthenhomogenizedinPBS(tissueweight(g):PBS(mL)volume=1:9)withaglasshomogenizeronice.
Tofurtherbreakdownthecells,youcansonicatethesuspensionwithanultrasoniccelldisrupterorsubjectittofreeze-thawcycles.
Thehomogenatesarethencentrifugedfor5-10minat5000*gtogetthesupernatant.
Celllysates:Foradherentcells,gentlywashthecellswithmoderateamountofpre-cooledPBSanddissociatethecellsusingtrypsin.
Collectthecellsuspensionintoacentrifugetubeandcentrifugefor5minat1000*g.
Discardthemediumandwashthecells3timeswithpre-cooledPBS.
Foreach1*106cells,add150-250μLofpre-cooledPBStokeepthecellssuspended.
Repeatthefreeze-thawprocessseveraltimesuntilthecellsarefullylysed.
Centrifugefor10minat1500*gat2-8℃.
Removethecellfragments,collectthesupernatanttocarryouttheassay.
Avoidrepeatedfreeze-thawcycles.
Cellculturesupernatantorotherbiologicalfluids:Centrifugesamplesfor20minat1000*gat2-8℃.
Collectthesupernatanttocarryouttheassay.
www.
elabscience.
cn132017年4月修订第七版Noteforsample:1.
Samplesshouldbeassayedwithin7dayswhenstoredat2-8℃,otherwisesamplesmustbedividedupandstoredat-20℃(≤1month)or-80℃(≤3months).
Avoidrepeatedfreeze-thawcycles.
2.
Pleasepredicttheconcentrationbeforeassaying.
Ifthesampleconcentrationisnotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.
3.
Ifthesampletypeisnotincludedinthemanual,apreliminaryexperimentissuggestedtoverifythevalidity.
4.
Ifalysisbufferisusedtopreparetissuehomogenatesorcellculturesupernatant,thereisapossibilityofcausingadeviationduetotheintroducedchemicalsubstance.
5.
Somerecombinantproteinmaynotbedetectedduetoamismatchingwiththecoatedantibodyordetectionantibody.
www.
elabscience.
cn142017年4月修订第七版Reagentpreparation1.
Bringallreagentstoroomtemperature(18~25℃)beforeuse.
FollowtheMicroplatereadermanualforset-upandpreheatitfor15minbeforeODmeasurement.
2.
WashBuffer:Dilute30mLofConcentratedWashBufferwith720mLofdeionizedordistilledwatertoprepare750mLofWashBuffer.
Note:ifcrystalshaveformedintheconcentrate,warmitina40℃waterbathandmixitgentlyuntilthecrystalshavecompletelydissolved3.
Standardworkingsolution:Centrifugethestandardat10,000*gfor1min.
Add1.
0mLofReferenceStandard&SampleDiluent,letitstandfor10minandinvertitgentlyseveraltimes.
Afteritdissolvesfully,mixitthoroughlywithapipette.
Thisreconstitutionproducesaworkingsolutionof10ng/mL.
Thenmakeserialdilutionsasneeded.
Therecommendeddilutiongradientisasfollows:10,5,2.
5,1.
25,0.
63,0.
32,0.
16,0ng/mL.
Dilutionmethod:Take7EPtubes,add500uLofReferenceStandard&SampleDiluenttoeachtube.
Pipette500uLofthe10ng/mLworkingsolutiontothefirsttubeandmixuptoproducea5ng/mLworkingsolution.
Pipette500uLofthesolutionfromtheformertubeintothelatteroneaccordingtothesesteps.
Theillustrationbelowisforreference.
Note:thelasttubeisregardedasablank.
Don'tpipettesolutionintoitfromtheformertube.
1052.
51.
250.
630.
320.
1604.
BiotinylatedDetectionAbworkingsolution:Calculatetherequiredamountbeforetheexperiment(100μL/well).
Inpreparation,slightlymorethancalculatedshouldbeprepared.
Centrifugethestocktubebeforeuse,dilutethe100*ConcentratedBiotinylatedDetectionAbto1*workingsolutionwithBiotinylatedDetectionAbDiluent.
5.
ConcentratedHRPConjugateworkingsolution:Calculatetherequiredamountbeforetheexperiment(100μL/well).
Inpreparation,slightlymorethancalculatedshouldbeprepared.
Dilutethe100*ConcentratedHRPConjugateto1*workingsolutionwithConcentratedHRPConjugateDiluent.
www.
elabscience.
cn152017年4月修订第七版Assayprocedure(Abriefassayprocedureisonthe20thpage)1.
AddtheStandardworkingsolutiontothefirsttwocolumns:Eachconcentrationofthesolutionisaddedinduplicate,toonewelleach,sidebyside(100uLforeachwell).
Addthesamplestotheotherwells(100uLforeachwell).
Covertheplatewiththesealerprovidedinthekit.
Incubatefor90minat37℃.
Note:solutionsshouldbeaddedtothebottomofthemicroELISAplatewell,avoidtouchingtheinsidewallandcausingfoamingasmuchaspossible.
2.
Removetheliquidoutofeachwell,donotwash.
Immediatelyadd100μLofBiotinylatedDetectionAbworkingsolutiontoeachwell.
CoverwiththePlatesealer.
Gentlymixup.
Incubatefor1hourat37°C.
3.
Aspirateordecantthesolutionfromeachwell,add350uLofwashbuffertoeachwell.
Soakfor1~2minandaspirateordecantthesolutionfromeachwellandpatitdryagainstcleanabsorbentpaper.
Repeatthiswashstep3times.
Note:amicroplatewashercanbeusedinthisstepandotherwashsteps.
4.
Add100μLofHRPConjugateworkingsolutiontoeachwell.
CoverwiththePlatesealer.
Incubatefor30minat37°C.
5.
Aspirateordecantthesolutionfromeachwell,repeatthewashprocessforfivetimesasconductedinstep3.
6.
Add90μLofSubstrateReagenttoeachwell.
Coverwithanewplatesealer.
Incubateforabout15minat37°C.
Protecttheplatefromlight.
Note:thereactiontimecanbeshortenedorextendedaccordingtotheactualcolorchange,butnotmorethan30min.
7.
Add50μLofStopSolutiontoeachwell.
Note:Addingthestopsolutionshouldbedoneinthesameorderasthesubstratesolution.
8.
Determinetheopticaldensity(ODvalue)ofeachwellatoncewithamicro-platereadersetto450nm.
www.
elabscience.
cn162017年4月修订第七版CalculationofresultsAveragetheduplicatereadingsforeachstandardandsamples,thensubtracttheaveragezerostandardopticaldensity.
Plotafour-parameterlogisticcurveonlog-loggraphpaper,withstandardconcentrationonthex-axisandODvaluesonthey-axis.
Ifthesampleshavebeendiluted,theconcentrationcalculatedfromthestandardcurvemustbemultipliedbythedilutionfactor.
IftheODofthesamplesurpassestheupperlimitofthestandardcurve,youshouldre-testitwithanappropriatedilution.
Theactualconcentrationisthecalculatedconcentrationmultipliedbythedilutionfactor.
TypicaldataAstheODvaluesofthestandardcurvemayvaryaccordingtotheconditionsoftheactualassayperformance(e.
g.
operator,pipettingtechnique,washingtechniqueortemperatureeffects),theoperatorshouldestablishastandardcurveforeachtest.
Typicalstandardcurveanddataisprovidedbelowforreferenceonly.
Concentration(ng/mL)1052.
51.
250.
630.
320.
160OD2.
4891.
7651.
0570.
5180.
2690.
1790.
1220.
065CorrectedOD2.
4241.
70.
9920.
4530.
2040.
1140.
057-www.
elabscience.
cn172017年4月修订第七版PrecisionIntra-assayPrecision(Precisionwithinanassay):3sampleswithlow,midrangeandhighlevelHumanANGⅡR1weretested20timesononeplate,respectively.
Inter-assayPrecision(Precisionbetweenassays):3sampleswithlow,midrangeandhighlevelHumanANGⅡR1weretestedon3differentplates,20replicatesineachplate.
Intra-assayPrecisionInter-assayPrecisionSample123123n202020202020Mean(ng/mL)0.
521.
034.
330.
531.
074.
76Standarddeviation0.
030.
060.
160.
030.
060.
15CV(%)5.
775.
833.
705.
665.
613.
15RecoveryTherecoveryofHumanANGⅡR1spikedatthreedifferentlevelsinsamplesthroughouttherangeoftheassaywasevaluatedinvariousmatrices.
SampleTypeRange(%)AverageRecovery(%)Serum(n=5)90-10798EDTAplasma(n=5)95-106100Cellculturemedia(n=5)96-111101LinearitySampleswerespikedwithhighconcentrationsofHumanANGⅡR1anddilutedwithReferenceStandard&SampleDiluenttoproducesampleswithvalueswithintherangeoftheassay.
Serum(n=5)EDTAplasma(n=5)Cellculturemedia(n=5)1:2Range(%)86-9888-10287-99Average(%)9294931:4Range(%)88-9986-9884-98Average(%)9493901:8Range(%)94-10685-9886-97Average(%)9991921:16Range(%)91-10383-9988-102Average(%)969095www.
elabscience.
cn182017年4月修订第七版TroubleshootingProblemCausesSolutionsPoorstandardcurveInaccuratepipettingCheckpipettes.
ImproperstandarddilutionEnsurebrieflyspinthevialofstandardanddissolvethepowderthoroughlybygentlemixing.
WellsarenotcompletelyaspiratedCompletelyaspiratewellsinbetweensteps.
LowsignalInsufficientincubationtimeEnsuresufficientincubationtime.
IncorrectassaytemperatureUserecommendedincubationtemperature.
Bringsubstratetoroomtemperaturebeforeuse.
InadequatereagentvolumesCheckpipettesandensurecorrectpreparation.
ImproperdilutionHRPconjugateinactiveorTMBfailureMixHRPconjugateandTMB,rapidcoloring.
DeepcolorbutlowvaluePlatereadersettingisnotoptimalVerifythewavelengthandfiltersettingontheMicroplatereader.
OpentheMicroplateReaderaheadtopre-heat.
LargeCVInaccuratepipettingCheckpipettes.
HighbackgroundConcentrationoftargetproteinistoohighUserecommendeddilutionfactor.
PlateisinsufficientlywashedReviewthemanualforproperwash.
Ifusingaplatewasher,checkthatallportsareunobstructed.
ContaminatedwashbufferPreparefreshwashbuffer.
LowsensitivityImproperstorageoftheELISAkitAllthereagentsshouldbestoredaccordingtotheinstructions.
StopsolutionisnotaddedStopsolutionshouldbeaddedtoeachwellbeforemeasurement.
www.
elabscience.
cn192017年4月修订第七版SUMMARY1.
Add100μLstandardorsampletoeachwell.
Incubatefor90minat37°C.
2.
Removetheliquid.
Add100μLBiotinylatedDetectionAb.
Incubatefor1hourat37°C.
3.
Aspirateandwash3times.
4.
Add100μLHRPConjugate.
Incubatefor30minat37°C.
5.
Aspirateandwash5times.
6.
Add90μLSubstrateReagent.
Incubatefor15minat37°C.
7.
Add50μLStopSolution.
Readat450nmimmediately.
8.
Calculationofresults.
Declaration1.
Limitedbycurrentconditionsandscientifictechnology,wecan'tconductcomprehensiveidentificationandanalysisonalltherawmaterialprovided.
Sotheremightbesomequalitativeandtechnicalrisksforusersusingthekit.
2.
Thefinalexperimentalresultswillbecloselyrelatedtothevalidityofproducts,operationalskillsoftheoperatorsandtheexperimentalenvironments.
Pleasemakesurethatsufficientsamplesareavailable.
3.
Togetthebestresults,pleaseonlyusethereagentssuppliedbythemanufacturerandstrictlycomplywiththeinstructions!
4.
Incorrectresultsmayoccurbecauseofincorrectoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingsoftheMicro-platereader.
Pleasereadtheinstructionscarefullyandadjusttheinstrumentpriortotheexperiment.
5.
Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.
Inordertogetreproducibleresults,theoperationofeverystepintheassayshouldbecontrolled.
6.
EverykithasstrictlypassedQCtest.
However,resultsfromendusersmightbeinconsistentwithourdataduetosomevariablessuchastransportationconditions,differentlabequipments,andsoon.
Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromtheabovereasons,too.
PoweredbyTCPDF(www.
tcpdf.
org)Copyright2017-2018ElabscienceBiotechnologyCo.
,Ltd.
AllRightsReserved