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FishVTGELISAKitForthequantitativeinvitrodeterminationofFishVitellogeninconcentrationsinbodyfluid-celiacfluid-tissuehomogenates-otherbiologicalfluidsFORLABORATORYRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.ELISAENZYMElinkEDIMMUNOSORBENTASSAYINTENDEDUSEANDTESTPRINCIPLEThisVTGELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofVTGinthesample，thisVTGELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusVTGconcentration.TheconcentrationofVTGinthesamplesisthendeterminedbycomparingtheO.D。ofthesamplestothestandardcurve.SAMPLECOLLECTIONANDSTORAGESSerum-Useaserumseparatortubeandallowsamplestoclotfortwohoursatroomtemperatureorovernightat4℃beforecentrifugationfor20minutesatapproximately1000×g.Assayfreshlypreparedserumimmediatelyorstoresamplesinaliquotat -20℃或-80℃forlateruse.Avoidrepeatedfreeze / thawcycles.Plasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gat2-8℃within30minutesofcollection.Removeplasmaandassayimmediatelyorstoresamplesinaliquotat -20℃或-80℃forlateruse.Avoidrepeatedfreeze / thawcycles。 Tissuehomogenates-Forgeneralinformation，hemolysisbloodmayaffecttheresult，soyoushouldrinsethetissueswithice-coldPBS（0.01M，pH值= 7.4）toremoveexcessbloodthoroughly.TissuepiecesshouldbeweighedandthenmincedtosmallpieceswhichwillbehomogenizedinPBS（thevolumedependsontheweightofthetissue。9mLPBSwouldbeappropriateto1gramtissuepieces.SomeproteaseinhibitorisrecommendedtoaddintothePBS）withaglasshomogenizeronice.Tofurtherbreakthecells，youcansonicatethesuspensionwithanultrasoniccelldisrupterorsubjectittofreeze-thawcycles.Thehomogenatesarethencentrifugatedfor5minutesat5000×gtogetthesupernate.Cellculturesupernatesandotherbiologicalfluids-Centrifugesamplesfor20minutesat1000×g.Removeparticulatesandassayimmediatelyorstoresamplesinaliquotat -20℃或-80℃forlateruse.Avoidrepeatedfreeze / thawcycles.Note：Thesamplesshoulebecentrifugateddequatelyandnohemolysisorgranulewasallowed.MATERIALSREQUIREDBUTNOTSUPPLIED1.37℃incubator2.Standardmicroplatereadercapableofmeasuringabsorbanceat450nm3.Precisionpipettes ，disposablepipettetipsandAbsorbentpaper4.DistilledordeionizedwaterREAGENTSPROVIDEDAllreagentsprovidedarestoredat2-8°C.Refertotheexpirationdateonthelabel。Name96determinations48determinationsMICROTITERPLATE8 * 12strips8 * 6stripsSTANDARD（6vial）0.3毫升/ vial0.3ml / vialSAMPLEDILUENT6.0ml3.0mlENZYMECONJUGATE10.0ml5.0mlWASHSOLUTION25ml15mlSUBSTRATEA6.0ml3.0mlSUBSTRATEB6.0ml3.0mlSTOPSOLUTION6.0ml3.0mlClosureplatemembrane22Usermanual11Sealedbags11Note：1.Standardconcentrationwasfollowedby：480,240,120,60,30,15ng / mL的.2.Ifsamplesgeneratevalueshigherthanthehigheststandard，pleasedilutethesampleswithSampleDiluentandrepeattheassay.PRECAUTIONSDonotsubstitutereagentsfromonekitlottoanother.Standard，conjugateandmicrotiterplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.Allowkitreagentsandmaterialstoreachroomtemperature（20-25℃）beforeuse.Donotusewaterbathstothawsamplesorreagents.Donotusekitcomponentsbeyondtheirexpirationdate.Useonlydeionizedordistilledwatertodilutereagents。Donotremovemicrotiterplatefromthestoragebaguntilneeded.Unusedstripsshouldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.Usefreshdisposablepipettetipsforeachtransfertoavoidcontamination.Donotmixacidandsodiumhypochloritesolutions.Serumandplasmashouldbehandledaspotentiallyhazardousandcapableoftransmittingdisease.Disposableglovesmustbewornduringtheassayprocedure，sincenoknowntestmethodcanoffercompleteassurancethatproductsderivedfromRatbloodwillnottransmitinfectiousagents.Therefore，allbloodderivativesshouldbeconsideredpotentiallyinfectiousandgoodlaboratorypracticesshouldbefollowed.Allsamplesshouldbedisposedofinamannerthatwillinactivateviruses.LiquidWaste：Addsodiumhypochloritetoafinalconcentrationof1.0％.Thewasteshouldbeallowedtostandforaminimumof30minutestoinactivatethevirusesbeforedisposal.SubstrateSolutioniseasilycontaminated.Ifbluishpriortouse，donotuse.SubstrateBcontain20％丙酮，keepthisreagentawayfromsourcesofheatorflame.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature（20-25℃）.REAGENTPREPARATIONANDSTORAGEWashSolution（1X）-Dilute1volumeofWashsolution（20X）with19volumesofdeionizedordistilledwater.WashSolutionisstablefor1monthat2-8°C.ASSAYPROCEDURE1.Prepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicrotiterplate.2.Add50μlofStandardorSampletotheappropriatewells.Blankwelldoesn'taddanyting.3。 Add100μlofEnzymeconjugatetostandardwellsandsamplewellsexcepttheblankwell，coverwithanadhesivestripandincubatefor60minutesat37°C.4.WashtheMicrotiterPlate4times.ManualWashing-Removeincubationmixturebyaspiratingcontentsoftheplateintoasinkorproperwastecontainer.Usingasquirtbottle，filleachwellcompletelywithWashSolution（1X），thenaspiratecontentsoftheplateintoasinkorproperwastecontainer.Repeatthisprocedureforatotaloffourtimes.Afterfinalwash，invertplate，andblotdrybyhittingplateontoabsorbentpaperorpapertowelsuntilnomoistureappears.Note：Holdthesidesoftheplateframefirmlywhenwashingtheplatetoassurethatallstripsremainsecurelyinframe.AutomatedWashing-Aspirateallwells，thenwashplatesfourtimesusingWashBuffer（1X）.Alwaysadjustyourwashertoaspirateasmuchliquidaspossibleandsetfillvolumeat350μL/孔/ wash.Afterfinalwash，invertplate，andblotdrybyhittingplateontoabsorbentpaperorpapertowelsuntilnomoistureappears.5.AddSubstrateA50μlandSubstrateB50μltoeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight.6.Add50μlStopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyellow .Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform，gentlytaptheplatetoensurethoroughmixing.7.ReadtheOpticalDensity（OD）at450nmusingamicrotiterplatereaderwithin15minutes.CALCULATIONOFRESULTSThisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D。（450nm处）obtainedforeachofthesixstandardconcentrationsonthevertical（X）axisversusthecorrespondingconcentrationonthehorizontal（Y）axis.First，calculatethemeanO.D.valueforeachstandardandsample.AllO.D.Valuesaresubtractedbythemeanvalueofthebalnkwellbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperorstatisticalsoftware.Todeterminetheamountineachsample，firstlocatetheO.D .valueontheY-axisandextendahorizontallinetothestandardcurve.Atthepointofintersection，drawaverticallinetotheX-axisandreadthecorrespondingconcentration.Anyvariationinoperator，pipettingandwashingtechnique，incubationtimeortemperature，andkitagecancausevariationinresult.Eachusershouldobtaintheirownstandardcurve.Intra-assayCV(%)islessthan10%andInter-assayCV(%)islessthan15%.Assayrange:15ng/mL480ng/mL.7.Sensitivity:TheminimumdetectabledoseofFishVTGistypicallylessthan1.0ng/mL.8.Cross-reactivity:ThisassayrecognizesrecombinantandnaturalFishVTG.Nosignificantcross-reactivityorinterferencewasobserved.9.Storage:2-8℃(Usefrequently);sixmonths(-20℃)。10.StandardcurveFORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!鱼卵黄蛋白原（VTG）试剂盒（ELISA）使用说明书l本试剂盒用于体外定量检测体液、腔液、组织匀浆及相关液体样本中鱼卵黄蛋白原（VTG）的含量。l有效期：6个月l保存条件：2-8℃实验原理试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。往预先包被鱼卵黄蛋白原（VTG）捕获抗体的包被微孔中，依次加入标本、标准品、HRP标记的检测抗体，经过温育并彻底洗涤。用底物TMB显色，TMB在过氧化物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鱼卵黄蛋白原（VTG）呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。样本处理及要求1.血清：将收集于血清分离管的全血标本在室温放置2小时或4℃过夜，然后1000×g离心20分钟，取上清即可，或将上清置于-20℃或-80℃保存，但应避免反复冻融。2.血浆：用EDTA或肝素作为抗凝剂采集标本，并将标本在采集后的30分钟内于2-8℃1000×g离心15分钟，取上清即可检测，或将上清置于-20℃或-80℃保存，但应避免反复冻融。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（匀浆中裂解的红细胞会影响测量结果），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，于冰上充分研磨。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。最后将匀浆液于5000×g离心5~10分钟，取上清检测。4.细胞培养物上清或其它生物标本：请1000×g离心20分钟，取上清即可检测，或将上清置于-20℃或-80℃保存，但应避免反复冻融。注：标本溶血会影响最后检测结果，因此溶血标本不宜进行此项检测。需要而未提供的试剂和器材酶标仪（450nm）高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL37℃恒温箱蒸馏水或去离子水试剂盒组成名称96孔配置48孔配置备注微孔酶标板8孔×12条8孔×6条无标准品0.3mL*6管0.3mL*6管无样本稀释液6mL3mL无检测抗体-HRP10mL5mL无20×洗涤缓冲液25mL15mL按说明书进行稀释底物A6mL3mL无底物B6mL3mL无终止液6mL3mL无封板膜2张2张无说明书1份1份无自封袋1个1个无备注：1.标准品浓度依次为：480、240、120、60、30、15ng/mL2.经过大量正常标本检验，标本的正常浓度值均在试剂盒提供的检测范围内，实验过程中直接取50μL样本上样即可。当有部分样本值超过最大标准品浓度时，可用样本稀释液将标本进行适当稀释后再进行实验。注意事项严格按照规定的时间和温度进行温育以保证准确结果。所有试剂都必须在使用前达到室温20-25℃。使用后立即冷藏保存试剂。洗板不正确可以导致不准确的结果。在加入底物前确保尽量吸干孔内液体。温育过程中不要让微孔干燥掉。消除板底残留的液体和手指印，否则影响OD值。底物显色液应呈无色或很浅的颜色，已经变蓝的底物液不能使用。避免试剂和标本的交叉污染以免造成错误结果。在储存和温育时避免强光直接照射。平衡至室温后再打开密封袋以防水滴凝聚在冷板条上。任何反应试剂不能接触漂白溶剂或漂白溶剂所散发的强烈气体。任何漂白成分都会破坏试剂盒中反应试剂的生物活性。不能使用过期产品。如果可能传播疾病，所有的样品都应管理好，按照规定的程序处理样品和检测装置。试剂准备试剂盒从冷藏环境中取出应在室温平衡后方可使用。20×洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份20×洗涤缓冲液加19份蒸馏水。操作步骤从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；样本孔中加入待测样本50μL；空白孔不加。除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。弃去液体，吸水纸上拍干，每孔加满洗涤液（350μL），静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机洗板）。每孔加入底物A、B各50μL，37℃避光孵育15min。每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。实验结果计算以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，在坐标纸上或用相关软件绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出样品的浓度。试剂盒性能检测范围：15ng/mL480ng/mL。灵敏度：最低检测浓度小于1.0ng/mL。特异性：不与其它可溶性结构类似物交叉反应。重复性：板内变异系数小于10%，板间变异系数小于15%。
鱼卵黄蛋白原（VTG）ELISA试剂盒说明书
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