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首页 猪血管活性肠肽VIP酶联免疫分析ELISA

猪血管活性肠肽VIP酶联免疫分析ELISA.doc

猪血管活性肠肽VIP酶联免疫分析ELISA

Nancy瑞敏
2019-06-24 0人阅读 举报 0 0 暂无简介

简介:本文档为《猪血管活性肠肽VIP酶联免疫分析ELISAdoc》,可适用于农林牧渔领域

猪血管活性肠肽(VIP)酶联免疫分析(ELISA)试剂盒使用说明书本试剂仅供研究使用   目的:本试剂盒用于测定猪血清血浆细胞上清及相关液体样本中血管活性肠肽(VIP)的含量。实验原理:本试剂盒应用双抗体夹心法测定标本中猪血管活性肠肽(VIP)水平。用纯化的猪血管活性肠肽(VIP)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血管活性肠肽(VIP)再与HRP标记的血管活性肠肽(VIP)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最终的黄色。颜色的深浅和样品中的血管活性肠肽(VIP)呈正相关。用酶标仪在nm波长下测定吸光度(OD值)通过标准曲线计算样品中猪血管活性肠肽(VIP)浓度。试剂盒组成:试剂盒组成孔配置孔配置保存说明书份份 封板膜片()片() 密封袋个个 酶标包被板××℃保存标准品:ngLml×瓶ml×瓶℃保存标准品稀释液ml×瓶ml×瓶℃保存酶标试剂ml×瓶ml×瓶℃保存样品稀释液ml×瓶ml×瓶℃保存显色剂A液ml×瓶ml×瓶℃保存显色剂B液ml×瓶ml×瓶℃保存终止液ml×瓶ml×瓶℃保存浓缩洗涤液(ml×倍)×瓶(ml×倍)×瓶℃保存    样本处理及要求:血清:室温血液自然凝固分钟离心分钟左右(转分)。仔细收集上清保存过程中如出现沉淀应再次离心。血浆:应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂混合分钟后离心分钟左右(转分)。仔细收集上清保存过程中如有沉淀形成应该再次离心。尿液:用无菌管收集离心分钟左右(转分)。仔细收集上清保存过程中如有沉淀形成应再次离心。胸腹水、脑脊液参照实行。细胞培养上清:检测分泌性的成份时用无菌管收集。离心分钟左右(转分)。仔细收集上清。检测细胞内的成份时用PBS(PH)稀释细胞悬液细胞浓度达到万ml左右。通过反复冻融以使细胞破坏并放出细胞内成份。离心分钟左右(转分)。仔细收集上清。保存过程中如有沉淀形成应再次离心。组织标本:切割标本后称取重量。加入一定量的PBSPH。用液氮迅速冷冻保存备用。标本融化后仍然保持℃的温度。加入一定量的PBS(PH)用手工或匀浆器将标本匀浆充分。离心分钟左右(转分)。仔细收集上清。分装后一份待检测其余冷冻备用。标本采集后尽早进行提取提取按相关文献进行提取后应尽快进行实验。若不能马上进行试验可将标本放于℃保存但应避免反复冻融不能检测含NaN的样品因NaN抑制辣根过氧化物酶的(HRP)活性。操作步骤标准品的稀释与加样:在酶标包被板上设标准品孔孔在第一、第二孔中分别加标准品μl然后在第一、第二孔中加标准品稀释液μl混匀然后从第一孔、第二孔中各取μl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μl混匀然后在第三孔和第四孔中先各取μl弃掉再各取μl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ul混匀混匀后从第五、第六孔中各取μl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μl混匀后从第七、第八孔中分别取μl加到第九、第十孔中再在第九第十孔分别加标准品稀释液μl混匀后从第九第十孔中各取μl弃掉。(稀释后各孔加样量都为μl浓度分别为ngLngLngLngLngL)。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μl然后再加待测样品μl(样品最终稀释度为倍)。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。温育:用封板膜封板后置℃温育分钟。配液:将(T的倍)倍浓缩洗涤液用蒸馏水(T的倍)倍稀释后备用。洗涤:小心揭掉封板膜弃去液体甩干每孔加满洗涤液静置秒后弃去如此重复次拍干。加酶:每孔加入酶标试剂μl空白孔除外。温育:操作同。洗涤:操作同。显色:每孔先加入显色剂Aμl再加入显色剂Bμl轻轻震荡混匀℃避光显色分钟终止:每孔加终止液μl终止反应(此时蓝色立转黄色)。测定:以空白空调零nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后分钟以内进行。注意事项:.试剂盒从冷藏环境中取出应在室温平衡分钟后方可使用酶标包被板开封后如未用完板条应装入密封袋中保存。.浓洗涤液可能会有结晶析出稀释时可在水浴中加温助溶洗涤时不影响结果。.各步加样均应使用加样器并经常校对其准确性以避免试验误差。一次加样时间最好控制在分钟内如标本数量多推荐使用排枪加样。.请每次测定的同时做标准曲线最好做复孔。如标本中待测物质含量过高(样本OD值大于标准品孔第一孔的OD值)请先用样品稀释液稀释一定倍数(n倍)后再测定计算时请最后乘以总稀释倍数(×n×)。.封板膜只限一次性使用以避免交叉污染。.底物请避光保存。.严格按照说明书的操作进行试验结果判定必须以酶标仪读数为准.所有样品洗涤液和各种废弃物都应按传染物处理。.本试剂不同批号组分不得混用。如与英文说明书有异以英文说明书为准。计算:以标准物的浓度为横坐标OD值为纵坐标  在坐标纸上绘出标准曲线根据样品的OD   值由标准曲线查出相应的浓度再乘以稀释   倍数或用标准物的浓度与OD值计算出标   准曲线的直线回归方程式将样品的OD值   代入方程式计算出样品浓度再乘以稀释   倍数即为样品的实际浓度。         (此图仅供参考)试剂盒性能:样品线性回归与预期浓度相关系数R值为以上。批内与批见应分别小于和检测范围:                       ngLngL                    保存条件及有效期:试剂盒保存:℃。.有效期:个月FORRESEARCHUSEONLYPorcineVasoactiveIntestinalPeptide DrugNamesGenericName:PorcineVasoactiveIntestinalPeptide(VIP)ELISAKitPurposeThiskitallowsforthedeterminationofVIPconcentrationsinPorcineserum,bloodplasma,andotherbiologicalfluidsPrincipleoftheassayThekitassayPorcineVIPlevelinthesampleusePurifiedPorcineVIPantibodytocoatmicrotiterplatewells,makesolidphaseantibody,thenaddVIPtowells,CombinedVIPantibodywhichWithHRPlabeled,becomeantibodyantigenenzymeantibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzymecatalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthofnmTheconcentrationofVIPinthesamplesisthendeterminedbycomparingtheODofthesamplestothestandardcurveMaterialsprovidedwiththekitMaterialsprovidedwiththekitdeterminationsdeterminationsStorageUsermanual Closureplatemembrane Sealedbags Microelisastripplate℃Standard:ngLml×bottleml×bottle℃Standarddiluentml×bottleml×bottle℃HRPConjugatereagentml×bottleml×bottle℃Samplediluentml×bottleml×bottle℃ChromogenSolutionAml×bottleml×bottle℃ChromogenSolutionBml×bottleml×bottle℃StopSolutionml×bottleml×bottle℃washsolution(ml×fold)×bottle(ml×fold)×bottle℃    Specimenrequirementsserumcoagulationatroomtemperatureminscentrifugationminatthespeedofrpmremovesupernatant,Ifprecipitationappeared,CentrifugalagainplasmausesuitedEDTAorcitrateorasananticoagulant,mixmins,centrifugationminatthespeedofrpmremovesupernatant,Ifprecipitationappeared,CentrifugalagainUrinecollectsueasterilecontainer,centrifugationminatthespeedofrpmremovesupernatant,Ifprecipitationappeared,CentrifugalagainTheOperationofHydrothoraxandcerebrospinalfluidReferencetoitcellculturesupernatantdetectsecretorycomponents,collectsueasterilecontainer,centrifugationminatthespeedofrpmremovesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPBS(PH),Cellconcentrationreachedmillionml,repeatedfreezethawcycles,damagecellsandreleaseofintracellularcomponents,centrifugationminatthespeedofrpmremovesupernatant,Ifprecipitationappeared,CentrifugalagainTissuesamplesAftercuttingsamples,checktheweight,addPBS(PH),Rapidlyfrozenwithliquidnitrogen,maintainsamplesat℃aftermelting,addPBS(PH),HomogenizedbyhandorGrinders,centrifugationminatthespeedofrpmremovesupernatantextractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextractionIfitcan’t,specimencanbekeptin℃topreserve,AvoidrepeatedfreezethawcyclesCan’tdetectthesamplewhichcontainNaN,becauseNaNinhibitsHRPactiveAssayprocedureDiluteandaddsampletoStandard:setStandardwellsontheELISAplatescoated,addStandardμltothefirstandthesecondwell,thenaddStandarddilutionμltothefirstandthesecondwell,mixtakeoutμlformthefirstandthesecondwellthenaddittothethirdandtheforthwellseparatelythenaddStandarddilutionμltothethirdandtheforthwell,mixthentakeoutμlfromthethirdandtheforthwelldiscard,addμltothefifthandthesixthwell,thenaddStandarddilutionμltothefifthandthesixthwell,mixtakeoutμlfromthefifthandthesixthwellandaddtotheseventhandtheeighthwell,thenaddStandarddilutionμltotheseventhandtheeighthwell,mixtakeoutμlfromtheseventhandtheeighthwellandaddtotheninthandthetenthwell,addStandarddilutionμltotheninthandthetenthwell,mix,takeoutμlfromtheninthandthetenthwelldiscard(addSampleμltoeachwellafterDiluting,(density:ngLngLngLngLngL)addsample:Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandHRPConjugatereagent,othereachstepoperationissame)testingsamplewelladdSampledilutionμltotestingsamplewell,thenaddtestingsampleμl(samplefinaldilutionisfold),addsampletowells,don’ttouchthewellwallasfaraspossible,andGentlymixIncubate:AfterclosingplatewithClosureplatemembrane,incubateforminat℃Configurateliquid:fold(orfold)washsolutiondilutedfold(orfold)withdistilledwaterandreservewashing:UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillforsthendrain,repeattimes,drybypataddenzyme:AddHRPConjugatereagentμltoeachwell,exceptblankwellincubate:Operationwithwashing:Operationwithcolor:AddChromogenSolutionAulandChromogenSolutionBtoeachwell,evadethelightpreservationforminat℃Stopthereaction:AddStopSolutionμltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor)assay:takeblankwellaszero,ReadabsorbanceatnmafterAddingStopSolutionandwithinminImportantnotesThekittakesoutfromtherefrigerationenvironmentshouldbebalancedminutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbagwashingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendiluteWashingdoesnotaffecttheresultaddSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerroraddsamplewithinmin,ifthenumberofsampleismuch,recommendtouseVolleyifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(nfold),Pleasediluenteandmultipliedbythedilutionfactor(×n×)Closureplatemembraneonlylimitsthedisposableuse,toavoidcrosscontaminationThesubstrateevadethelightpreservationPleaseaccordingtouseinstructionstrictly,ThetestresultdeterminationmusttakethemicrotiterplatereaderasastandardAllsamples,washingbufferandeachkindofrejectshouldaccordingtoinfectivematerialprocessDonotmixreagentswiththosefromotherlotsTakethestandarddensityasthehorizontal,theODvalueforthevertical,drawthestandardcurveongraphpaper,FindoutthecorrespondingdensityaccordingtothesampleODvaluebytheSamplecurve,multipliedbythedilutionmultiple,orcalculatethestraightlineregressionequationofthestandardcurvewiththestandarddensityandtheODvalue,withthesampleODvalueintheequation,calculatethesampledensity,multipliedbythedilutionfactor,theresultisthesampleactualdensityCalculate

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