大鼠淀粉酶AMS说明书模板.doc

资料内容仅供您学习参考，如有不当或者侵权，请联系改正或者删除。大鼠淀粉酶(AMS)酶联免疫分析(ELISA)试剂盒使用说明书齐一生物科技( 上海) 有限公司本试剂仅供研究使用目的: 本试剂盒用于测定大鼠血清, 血浆及相关液体样本中淀粉酶(AMS)的含量。实验原理: 本试剂盒应用双抗体夹心法测定标本中大鼠淀粉酶(AMS)水平。用纯化的大鼠淀粉酶(AMS)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淀粉酶(AMS), 再与HRP标记的淀粉酶(AMS)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淀粉酶(AMS)呈正相关。用酶标仪在450nm波长下测定吸光度( OD值) , 经过标准曲线计算样品中大鼠淀粉酶(AMS)浓度。试剂盒组成: 试剂盒组成48孔配置96孔配置保存说明书1份1份封板膜2片( 48) 2片( 96) 密封袋1个1个酶标包被板1481962-8保存标准品: 180mol/L0.5ml1瓶0.5ml1瓶2-8保存标准品稀释液1.5ml1瓶1.5ml1瓶2-8保存酶标试剂3ml1瓶6ml1瓶2-8保存样品稀释液3ml1瓶6ml1瓶2-8保存显色剂A液3ml1瓶6ml1瓶2-8保存显色剂B液3ml1瓶6ml1瓶2-8保存终止液3ml1瓶6ml1瓶2-8保存浓缩洗涤液( 20ml20倍) 1瓶( 20ml30倍) 1瓶2-8保存样本处理及要求: 1.血清: 室温血液自然凝固10-20分钟, 离心20分钟左右( -3000转/分) 。仔细收集上清, 保存过程中如出现沉淀, 应再次离心。2.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10-20分钟后, 离心20分钟左右( -3000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。3.尿液: 用无菌管收集, 离心20分钟左右( -3000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。4.细胞培养上清: 检测分泌性的成份时, 用无菌管收集。离心20分钟左右( -3000转/分) 。仔细收集上清。检测细胞内的成份时, 用PBS( PH7.2-7.4) 稀释细胞悬液, 细胞浓度达到100万/ml左右。经过重复冻融, 以使细胞破坏并放出细胞内成份。离心20分钟左右( -3000转/分) 。仔细收集上清。保存过程中如有沉淀形成, 应再次离心。5.组织标本: 切割标本后, 称取重量。加入一定量的PBS, PH7.4。用液氮迅速冷冻保存备用。标本融化后依然保持2-8的温度。加入一定量的PBS( PH7.4) , 用手工或匀浆器将标本匀浆充分。离心20分钟左右( -3000转/分) 。仔细收集上清。分装后一份待检测, 其余冷冻备用。6.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-20保存, 但应避免重复冻融.7.不能检测含NaN3的样品, 因NaN3抑制辣根过氧化物酶的( HRP) 活性。操作步骤: 1. 标准品的稀释与加样: 在酶标包被板上设标准品孔10孔, 在第一、 第二孔中分别加标准品100l, 然后在第一、 第二孔中加标准品稀释液50l, 混匀; 然后从第一孔、 第二孔中各取100l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50l, 混匀; 然后在第三孔和第四孔中先各取50l弃掉, 再各取50l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50ul, 混匀; 混匀后从第五、 第六孔中各取50l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50l, 混匀后从第七、 第八孔中分别取50l加到第九、 第十孔中, 再在第九第十孔分别加标准品稀释液50l, 混匀后从第九第十孔中各取50l弃掉。( 稀释后各孔加样量都为50l, 浓度分别为120mol/L, 80mol/L, 40mol/L, 20mol/L, 10mol/L) 。2. 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40l, 然后再加待测样品10l( 样品最终稀释度为5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。3. 温育: 用封板膜封板后置37温育30分钟。4. 配液: 将30( 48T的20倍) 倍浓缩洗涤液用蒸馏水30( 48T的20倍) 倍稀释后备用。5. 洗涤: 小心揭掉封板膜, 弃去液体, 甩干, 每孔加满洗涤液, 静置30秒后弃去, 如此重复5次, 拍干。6. 加酶: 每孔加入酶标试剂50l, 空白孔除外。7. 温育: 操作同3。8. 洗涤: 操作同5。9. 显色: 每孔先加入显色剂A50l, 再加入显色剂B50l, 轻轻震荡混匀, 37避光显色15分钟.10. 终止: 每孔加终止液50l, 终止反应( 此时蓝色立转黄色) 。11. 测定: 以空白空调零, 450nm波长依序测量各孔的吸光度( OD值) 。测定应在加终止液后15分钟以内进行。注意事项: 1 试剂盒从冷藏环境中取出应在室温平衡15-30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。2 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。3 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5分钟内, 如标本数量多, 推荐使用排枪加样。4 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( n5) 。5 封板膜只限一次性使用, 以避免交叉污染。6 底物请避光保存。7 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.8 所有样品, 洗涤液和各种废弃物都应按传染物处理。9 本试剂不同批号组分不得混用。10.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: 1.样品线性回归与预期浓度相关系数R值为0.92以上。2.批内与批见应分别小于9%和15%检测范围: 5mol/L-150mol/L保存条件及有效期: 1.试剂盒保存: ; 2-8。2有效期: 6个月FORRESEARCHUSEONLYRatAmylaseDrugNamesGenericName: RatAmylase(AMS)ELISAKit.PurposeThiskitallowsforthedeterminationofAMSconcentrationsinRatserum,bloodplasma,andotherbiologicalfluids.PrincipleoftheassayThekitassayRatAMSlevelinthesample, usePurifiedRatAMStocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddAMStowells,CombinedAMSantibodywhichWithHRPlabeled,becomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofAMSinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8Standard: 180mol/L0.5ml1bottle0.5ml1bottle2-8Standarddiluent1.5ml1bottle1.5ml1bottle2-8HRP-Conjugatereagent3ml1bottle6ml1bottle2-8Samplediluent3ml1bottle6ml1bottle2-8ChromogenSolutionA3ml1bottle6ml1bottle2-8ChromogenSolutionB3ml1bottle6ml1bottle2-8StopSolution3ml1bottle6ml1bottle2-8washsolution( 20ml20fold) 1bottle( 20ml30fold) 1bottle2-8Specimenrequirements1. serum-coagulationatroomtemperature10-20mins, centrifugation20-minatthespeedof -3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.2. plasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof -3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.3. Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof -3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.4. cellculturesupernatant-detectsecretorycomponents,collectsueasterilecontainer,centrifugation20-minatthespeedof -3000r.p.m.removesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPBS( PH7.2-7.4) ,Cellconcentrationreached1million/ml,repeatedfreeze-thawcycles,damagecellsandreleaseofintracellularcomponents,centrifugation20-minatthespeedof -3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.5. Tissuesamples-Aftercuttingsamples,checktheweight,addPBS( PH7.2-7.4) ,Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8aftermelting,addPBS( PH7.4) ,HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof -3000r.p.m.removesupernatant.6. extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcant,specimencanbekeptin-20topreserve,Avoidrepeatedfreeze-thawcycles.7. CantdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure1.DiluteandaddsampletoStandard:set10StandardwellsontheELISAplatescoated,addStandard100ltothefirstandthesecondwell,thenaddStandarddilution50ltothefirstandthesecondwell,mix;takeout100lformthefirstandthesecondwellthenaddittothethirdandtheforthwellseparately.thenaddStandarddilution50ltothethirdandtheforthwell,mix;thentakeout50lfromthethirdandtheforthwelldiscard,add50ltothefifthandthesixthwell,thenaddStandarddilution50ltothefifthandthesixthwell,mix;takeout50lfromthefifthandthesixthwellandaddtotheseventhandtheeighthwell,thenaddStandarddilution50ltotheseventhandtheeighthwell,mix;takeout50lfromtheseventhandtheeighthwellandaddtotheninthandthetenthwell,addStandarddilution50ltotheninthandthetenthwell,mix,takeout50lfromtheninthandthetenthwelldiscard(addSample50ltoeachwellafterDiluting,(density:120mol/L, 80mol/L, 40mol/L, 20mol/L, 10mol/L)2.addsample: Setblankwellsseparately(blankcomparisonwellsdontaddsampleandHRP-Conjugatereagent,othereachstepoperationissame).testingsamplewell.addSampledilution40ltotestingsamplewell,thenaddtestingsample10l(samplefinaldilutionis5-fold),addsampletowells,donttouchthewellwallasfaraspossible,andGentlymix.3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37.4.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwaterandreserve.5.washing: UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.6.addenzyme: AddHRP-Conjugatereagent50ltoeachwell,exceptblankwell.7.incubate: Operationwith3.8.washing: Operationwith5.9.color: AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat3710.Stopthereaction: AddStopSolution50ltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).11.assay: takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.Importantnotes1. Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.2. washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.3. addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5mins,ifthenumberofsampleismuch,recommendtouseVolley.4. ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.( n5) .5. Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.6. Thesubstrateevadethelightpreservation.7. Pleaseaccordingtouseinstructionstrictly,Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.8. Allsamples,washingbufferandeachkindofrejectshouldaccordingtoinfectivematerialprocess.9. Donotmixreagentswiththosefromotherlots.Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.CalculateThis chart for reference onlyAssayrange5mol/L-150mol/LStorageandvalidity1Storage: 2-8.2validity: sixmonths.