大鼠(Rat)25羥基維生素D3(25(OH)D3)

大鼠(Rat)25羥基維生素D3(25(0H)D3)ELISA檢測(cè)試劑盒使用說(shuō)明書檢測(cè)原理試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)先包被25羥基維生素D3（25（OH）D3）抗體的包被微孔中，依次加入血漿：EDTA、檸檬酸鹽或肝素抗凝。3000轉(zhuǎn)離心30分鐘取上清。細(xì)胞上清液：3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物。組織勻漿：將組織加入適量生理鹽水搗碎。3000轉(zhuǎn)離心10分鐘取上清。保存：如果樣本收集后不及時(shí)檢測(cè)，請(qǐng)按一次用量分裝，凍存于-20°C，避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍。標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體，經(jīng)過(guò)溫育并徹底洗滌。用底物自備物品TMB顯色，TMB在過(guò)氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下酶標(biāo)儀（450nm）轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的25羥基維生素D3高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL（25（OH）D3）呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），計(jì)算樣品濃度。樣品收集、處理及保存方法血清：使用不含熱原和內(nèi)毒素的試管，操作過(guò)程中避免任何細(xì)胞37C恒溫箱操作注意事項(xiàng)試劑盒保存在2-8C，使用前室溫平衡20分鐘。從冰箱取出的濃縮洗滌液會(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶完全溶解實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。沖液加19份的蒸餾水。濃度為0的SO號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白；按照說(shuō)明書操作時(shí)樣本已經(jīng)稀釋5倍，最終結(jié)果乘以5才是樣本實(shí)際濃度。嚴(yán)格按照說(shuō)明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。所有液體組分使用前充分搖勻。試劑盒組成名稱96孔配置48孔配置備注微孔酶標(biāo)板12孔x8條12孔x4條無(wú)標(biāo)準(zhǔn)品0.3mL*6管0.3mL*6管無(wú)樣本稀釋液6mL3mL無(wú)檢測(cè)抗體-HRP10mL5mL無(wú)20x洗滌緩沖液25mL15mL按說(shuō)明書進(jìn)行稀釋底物A6mL3mL無(wú)底物B6mL3mL無(wú)終止液6mL3mL無(wú)封板膜2張2張無(wú)說(shuō)明書1份1份無(wú)自封袋1個(gè)1個(gè)無(wú)注：標(biāo)準(zhǔn)品（S0-S5）濃度依次為：0、3、6、12、24、48ng/mL試劑的準(zhǔn)備20x洗滌緩沖液的稀釋：蒸餾水按1：20稀釋，即1份的20x洗滌緩洗板方法手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液，靜置1min后甩盡孔內(nèi)液體，在吸水紙上拍干，如此洗板5次。自動(dòng)洗板機(jī)：每孔注入洗液350ML，浸泡1min，洗板5次。操作步驟從室溫平衡20min后的鋁箔袋中取出所需板條，剩余板條用自封袋密封放回4°C。設(shè)置標(biāo)準(zhǔn)品孔和樣本孔,標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50mL；樣本孔先加待測(cè)樣本10pL，再加樣本稀釋液40ML；空白孔不加。除空白孔外，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過(guò)氧化物酶（HRP）標(biāo)記的檢測(cè)抗體100pL,用封板膜封住反應(yīng)孔，37C水浴鍋或恒溫箱溫育60min。棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1min，甩去

每孔加入底物A、B各50兒37°C避光孵育15min。每孔加入終止液50pL,15min內(nèi)，在450nm波長(zhǎng)處測(cè)定各孔的OD值。結(jié)果判斷繪制標(biāo)準(zhǔn)曲線：在Excel工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)本濃度值。standardsconcentration(X)OD值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣本濃度值。standardsconcentration(X)準(zhǔn)確性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值，大于等于0.9900。靈敏度：最低檢測(cè)濃度小于1.0ng/mL。特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。重復(fù)性：板內(nèi)、板間變異系數(shù)均小于15%。5?貯藏:2-8C，避光防潮保存。有效期：6個(gè)月免責(zé)聲明試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。嚴(yán)格按照說(shuō)明書操作，實(shí)驗(yàn)者違反說(shuō)明書操作，后果由實(shí)驗(yàn)者承擔(dān)。Note:Note:FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Rat25-DihydroxyvitaminD3(25(OH)D3/HVD3)ELISAKitinstructionIntendeduseThis25(OH)D3/HVD3ELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.Inordertomeasuretheconcentrationof25(OH)D3/HVD3in

freeze-thawcyclesPlasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor30minutesat3000xgat2-8°Cwithin30minutesofcollection.Storesamplesat-20Cor-80C.Avoidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20Cor-80CAvoidrepeatedfreeze-thawcycles.Thesamplesshoulebecentrifugateddequatelyandnohemolysisorgranulewasallowed.MaterialsrequiredbutnotsuppliedStandardmicroplatereader(450nm)2.PrecisionpipettesandDisposablepipettetips.2.PrecisionpipettesandDisposablepipettetips.thesample,this25(OH)D3/HVD3ELISAKitincludesasetofcalibrationstandards.Thecalibrationstandardsareassayedatthesametimeasthesamplesandallowthe

3. 37CincubatoroperatortoproduceastandardcurveofOpticalDensityversus25(OH)D3/HVD3

Precautionsconcentration.Theconcentrationof25(OH)D3/HVD3inthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.SamplecollectionandstoragesSerum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesatapproximately3000xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20Cor-80C.Avoidrepeated

Donotsubstitutereagentsfromonekittoanother.Standard,conjugateandmicroplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.Donotremovemicroplatefromthestoragebaguntilneeded.Unusedstripsshouldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.Mixallreagentsbeforeusing.

Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature(20-25°C)MaterialssuppliedName96determinations48determinationsMicroelisastripplate12*8strips12*4stripsStandard0.3ml*6tubes0.3ml*6tubesSampleDiluent6.0ml3.0mlHRP-Conjugatereagent10.0ml5.0ml20XWashsolution25ml15mlChromogenSolutionA6.0ml3.0mlChromogenSolutionB6.0ml3.0mlStopSolution6.0ml3.0mlClosureplatemembrane22Usermanual11Sealedbags11Note:Standard(SOfS5)concentrationwasfollowedby:0,3,6,12,24,48ng/mlReagentpreparation20xwashsolution:DilutewithDistilledordeionizedwater1:20.AssayprocedurePrepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.Addstandard:SetStandardwells,testingsamplewells.Addstandard50^1toAddSample:Addtestingsample10g1thenaddSampleDiluent40gltotestingsamplewell;Blankwelldoesn'taddanyting.Add100glofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincubatefor60minutesat37°C.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.WashbyfillingeachwellwithWashSolution(400卩l(xiāng))usingasquirtbottle,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.AddchromogensolutionA50g1andchromogensolutionB50g1toeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight.Add50glStopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.Calculationofresults1.Thisstandardcurveisusedtodeterminetheamountinanunknownsample.standardwell.standardwell.ThestandardcurveisgeneratedbyplottingtheaverageO.D.(450nm)obtainedforeachofthesixstandardconcentrationsonthevertical(Y)axisversusthecorrespondingconcentrationonthehorizontal(X)axis.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,aresubtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperor