人β淀粉樣蛋白142 (Aβ142)酶聯(lián)免疫檢測(cè)

人卩淀粉樣蛋白1-42(AP1-42)酶聯(lián)免疫檢測(cè)試劑盒使用說(shuō)明書(shū) 畫(huà)］^使用前仔細(xì)閱讀本說(shuō)明書(shū)。本酶聯(lián)免疫試劑盒是基于雙抗體夾心技術(shù)原理,來(lái)檢測(cè)人褪黑素(AP1-42)，只能用于研究用途，不得用于醫(yī)學(xué)診斷。用 途：用于人血清、血漿及相關(guān)液體樣本中褪黑素(AP1-42)測(cè)定。工作原理本試劑盒采用的是生物素雙抗體夾心酶聯(lián)免疫吸附法(ELISA)測(cè)定樣品中人褪黑素(AP1-42)水平。向預(yù)先包被了人褪黑素(AP1-42)單克隆抗體的酶標(biāo)孔中加入人褪黑素(Ap1-42)，溫育；溫育后，加入生物素標(biāo)記的抗褪黑素(AP1-42)抗體。再與鏈霉親和素-HRP結(jié)合，形成免疫復(fù)合物，再經(jīng)過(guò)溫育和洗滌,去除未結(jié)合的酶，然后加入底物A、B,產(chǎn)生藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺與樣品中人褪黑素(AP1-42)的濃度呈正相關(guān)。試劑盒組成試劑盒組成48孔配置96孔配置保存說(shuō)明書(shū)1份1份封板膜2片(48)2片(96)密封袋1個(gè)1個(gè)酶標(biāo)包被板1X481X962-8€保存標(biāo)準(zhǔn)品6400pg/ml0.5mlX1瓶0.5mlX1瓶2-8€保存標(biāo)準(zhǔn)品稀釋液3mlX1瓶6mlX1瓶2-8€保存鏈霉親和素-HRP3mlX1瓶6mlX1瓶2-8€保存生物素標(biāo)記的抗褪黑素(Ap1-42)抗體0.5mlX1瓶1mlX1瓶2-8€保存顯色劑A液3mlX1瓶6mlX1瓶2-8€保存顯色劑B液3mlX1瓶6mlX1瓶2-8€保存終止液3mlX1瓶6mlX1瓶2-8€保存濃縮洗滌液(20mlX20倍)X1瓶(20mlX30倍)X1瓶2-8€保存需要而未提供的試劑和器材37°C恒溫箱。標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀。精密移液器及一次性吸頭蒸餾水，一次性試管吸水紙注意事項(xiàng)從2-8C取出的試劑盒，在開(kāi)啟試劑盒之前要室溫平衡至少30分鐘。酶標(biāo)包被板開(kāi)封后如未用完，板條應(yīng)裝入密封袋中保存。各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差嚴(yán)格按照說(shuō)明書(shū)的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).為避免交叉污染，要避免重復(fù)使用手中的吸頭和封板膜。不用的其它試劑應(yīng)包裝好或蓋好。不同批號(hào)的試劑不要混用。保質(zhì)前使用。底物B對(duì)光敏感，避免長(zhǎng)時(shí)間暴露于光下。洗板方法手工洗板方法：甩掉酶標(biāo)板內(nèi)的液體；在實(shí)驗(yàn)臺(tái)上鋪墊幾層吸水紙，酶標(biāo)板朝下用力拍幾次；將稀釋后的洗滌液至少0.35ml注入孔內(nèi)，浸泡1-2分鐘。根據(jù)需要，重復(fù)此過(guò)程數(shù)次。自動(dòng)洗板：如果有自動(dòng)洗板機(jī)，應(yīng)在熟練使用后再用到正式實(shí)驗(yàn)過(guò)程中標(biāo)本要求不能檢測(cè)含NaN3的樣品，因NaN3抑制辣根過(guò)氧化物酶的（HRP）活性。標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20°C保存，但應(yīng)避免反復(fù)凍融。操作程序標(biāo)準(zhǔn)品的稀釋：（本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進(jìn)行稀釋。）3200pg/ml5號(hào)標(biāo)準(zhǔn)品120》l的原倍標(biāo)準(zhǔn)品加入120》l標(biāo)準(zhǔn)品稀釋液1600pg/ml4號(hào)標(biāo)準(zhǔn)品120》l的5號(hào)標(biāo)準(zhǔn)品加入120》l標(biāo)準(zhǔn)品稀釋液800pg/ml3號(hào)標(biāo)準(zhǔn)品120》l的4號(hào)標(biāo)準(zhǔn)品加入120》l標(biāo)準(zhǔn)品稀釋液400pg/ml2號(hào)標(biāo)準(zhǔn)品120》l的3號(hào)標(biāo)準(zhǔn)品加入120》l標(biāo)準(zhǔn)品稀釋液200pg/ml1號(hào)標(biāo)準(zhǔn)品120》l的2號(hào)標(biāo)準(zhǔn)品加入120》l標(biāo)準(zhǔn)品稀釋液根據(jù)代測(cè)樣品數(shù)量加上標(biāo)準(zhǔn)品的數(shù)量決定所需的板條數(shù)。每個(gè)標(biāo)準(zhǔn)品和空白孔建議做復(fù)孔。每個(gè)樣品根據(jù)自己的數(shù)量來(lái)定，能使用復(fù)孔的盡量做復(fù)孔。加樣：1）空白孔，空白對(duì)照孔不加樣品，生物素標(biāo)記的抗褪黑素（AB1-42）抗體，鏈霉親和素-HRP，只加顯色劑A&B和終止液，其余各步操作相同；2）標(biāo)準(zhǔn)品孔：加入標(biāo)準(zhǔn)品50ul,鏈霉親和素-HRP50ul（標(biāo)準(zhǔn)品中已事先整合好生物素抗體，故不加）;3）代測(cè)樣品孔：加入樣本40ul,然后各加入抗褪黑素（AB1-42）抗體10ul、鏈霉親和素-HRP50ul，蓋上封板膜，輕輕震蕩混勻，37C溫育60分鐘。配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用。洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復(fù)5次，拍干。顯色：每孔先加入顯色劑A50ul，再加入顯色劑B50M1，輕輕震蕩混勻，37C避光顯色15分鐘.終止：每孔加終止液50M1,終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。測(cè)定：以空白空調(diào)零，450nm波長(zhǎng)依序測(cè)量各孔的吸光度（OD值）。測(cè)定應(yīng)

在加終止液后10分鐘以內(nèi)進(jìn)行。根據(jù)標(biāo)準(zhǔn)品的濃度及對(duì)應(yīng)的OD值計(jì)算出標(biāo)準(zhǔn)曲線的直線回歸方程，再根據(jù)樣品的OD值在回歸方程上計(jì)算出對(duì)應(yīng)的樣品濃度。也可以使用各種應(yīng)用軟件來(lái)計(jì)算。操作程序總結(jié):檢測(cè)范圍：100pg/ml—3200pg/ml。保存：2-8°C。有效期：6個(gè)月（2-8C）。HumanAP1-42ELISAKitInstruetionThiskitisonlyforseientificresearch,andshallnotbeusedasaclinicaldiagnosisofuse.PurposeThiskitallowsforthedeterminationofA01-42concentrationsinHumanserum,cellculturesupernatant,andotherbiologicalfluids.PrincipleThekitassayHumanAp1-42levelinthesample，addHumanAp1-42antibodytomicrotiterplatewells,afterIncubating,addBiotinylatedanti-A01-42-antibody,thenCombinedStreptavidin-HRP,becomecomplex,afterIncubatingandwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolor,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofA01-42inthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8°CStandard：6400pg/ml0.5mlx1bottle0.5mlx1bottle2-8°CStandarddiluent3mlx1bottle6mlx1bottle2-8CStreptavidin-HRP3mlx1bottle6mlx1bottle2-8CBiotinylatedanti—A01-42-antibody0.5mlx1bottle1mlx1bottle2-8CChromogenSolutionA3mlx1bottle6mlx1bottle2-8CChromogenSolutionB3mlx1bottle6mlx1bottle2-8CStopSolution3mlx1bottle6mlx1bottle2-8Cwashsolution(20mlx20fold)xlbottle(20mlx30fold)x1bottle2-8CMaterialsrequiredbutnotsupplied37°CincubatorStandardmicroplatereader(45Onm)PrecisionpipettesandDisposablepipettetips.deionizedwater.DisposableTesttube6AbsorbentpaperImportantnotesThekittakesoutfromtherefrigerationenvironmentshouldbebalaneed15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.Pleaseaccordingtouseinstructionstrictly,Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.UsenewdisposalplasticpipettetipsandClosureplatemembraneforeachstandard,inordertoavoidcrosscontamination.Donotmixreagentswiththosefromotherlots.Thesubstrateevadethelightpreservation.SpecimenrequirementsextractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan't,specimencanbekeptin-20Ctopreserve,Avoidrepeatedfreeze-thawcycles.Can'tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.AssayprocedureDiluteandaddsample:DiluteOriginaldensityStandardasfollowtable:3200pg/ml5Standard120plOriginaldensityStandard+120plStandarddiluent1600pg/ml4Standard120pl5Standard+120plStandarddiluent800pg/ml3Standard120pl4Standard+120plStandarddiluent400pg/ml2Standard120pl3Standard+120plStandarddiluent200pg/ml1Standard120pl2Standard+120plStandarddiluentaccordingtestingSamplenumberstodefinehowmanywellsnedd,StandardandblanksuggestedDoholes.addsample：1)blankwells:(blankcomparisonwellsdon'taddsample,Biotinylatedanti-Ap1-42-antibodyandStreptavidin-HRP,othereachstepoperationissame);2)Standardwells:addStandard50plandStreptavidin-HRP50》l;3)testingSamplewells:addsample40》l,thenaddanti-A01-42-antibody10pl,Streptavidin-HRP50pl.closingplatewithClosureplatemembrane,incubatefor60minat37°C.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwaterandreserve.washing：UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.color:AddChromogenSolutionA50ulandChro