猪传染性胸膜肺炎放线杆菌TAPP酶联免疫分析ELISA试剂盒使用说明书.docx

猪传染性胸膜肺炎放线杆菌(TAPP)酶联免疫分析(E1.ISA)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，血浆及相关液体样本中传染性网膜肺炎放线杆菌(TAPP)水平。实验原理：本试剂盒采用双抗体夹心法测定血清或血浆中猪传染性胸膜肺炎放线杆菌(TAPP)。用纯化的猪传染性胸膜肺炎放线杆菌(TAPP)抗体包被微孔板，制成固相抗体，可与样品中传染性胸膜肺炎放线杆菌(TAPP)相结合，经洗涤除去未结合的抗原和其他成分后再与HRP标记的传染性胸膜肺炎放线杆菌(TAPP)抗体结合，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。用随标仪在45Onm波长下测定吸光度(OD值),与CUTOFF值相比较,从而判定标本中猪传染性胸膜肺炎放线杆菌(TAPP)的存在与否。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1X481X962-8保存阴性对照0.5mlXl瓶0.5mlXl瓶28保存阳性对照0.5mlXl瓶0.5mlXl瓶2-8C保存酶标试剂3ml×l瓶6ml×1瓶2-8保存样品稀释液3ml×1瓶6ml×1瓶2-8C保存显色剂A液3ml×l瓶6ml×l瓶2-8保存显色剂B液3ml×l瓶6ml×l瓶2-8保存终止液3mlXl瓶6mlXl瓶2-8保存浓缩洗涤液(20mlX20倍)Xl瓶(20mlX30倍)Xl瓶2-8C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(2000-3000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(2000-3000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(2000-3000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(2000-3000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IOO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20分钟左右（2000-3000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,PH7.4o用液氮迅速冷冻保存备用。标本融化后仍然保持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（2000-3000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°C保存，但应避免反复冻融.7 .不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酸的（HRP）活性。操作步骤：1 .编号：将样品对应微孔按序编号，每板应设阴性对照2孔、阳性对照2孔、空白对照1孔（空白对照孔不加样品及醐标试剂，其余各步操作相同）2 .加样：分别在阴、阳性对照孔中加入阴性对照、阳性对照501.然后在待测样品孔先加样品稀释液40l,然后再加待测样品101.加样将样品加于陶标板孔底部，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）倍浓缩洗涤液加蒸储水至600ml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 .加醐：每孔加入随标试剂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）。I1.测定：以空白空调零，45Onm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。结果判定：试验有效性：阳性对照孔平均值21.00;阴性对照平均值0.15临界值（CUTOFF）计算：临界值=阴性对照孔平均值+0.10（阴性对照平均值低于0.05按0.05计算）阴性判定：样品OD值临界值（CUTOFF）者为传染性胸膜肺炎放线杆菌（TAPP）阴性阳性判定：样品OD值<临界值（CUTOFF）者为传染性胸膜肺炎放线杆菌（TAPP）阳性注意事项1 .操作严格按照说明书进行，木试剂不同批号组分不得混用。2 .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，酣标包被板开封后如未用完，板条应装入密封袋中保存。3 .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。4 .封板膜只限一次性使用，以避免交叉污染。5 .底物请避光保存。6 .试验结果判定必须以前标仪读数为准，使用双波长检测时，参考波长为630nm7 .所有样品，洗涤液和各种废弃物都应按传染物处理。终止液为2M的硫酸，使用时必须注意安全。保存条件及有效期1 .试剂盒保存：;2-8oC<,2 .有效期：6个月FORRESEARCHUSEON1.YPorcineTransmissibleActinobacillusPleuropneumoniaeDrugNamesGenericName：PorcineTransmissibleActinobacilluspleuropneumoniae(TAPP)E1.ISAKit.PurposeThiskitallowsforthedeterminationofTAPPinPorcineserum,andotherbiologicalfluids.PrincipleoftheassayThekitassayTAPPlevelinthesample,usePurifiedTAPPantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddTAPPtowells,CombinedWithTAPP,afterwashingandremovingnon-mbinativeantigenandothercomponents,thenCombinedIAPPantibodywhichWithHRPlabeled,afterwashingCompletely,AddTMBsubstrateSoIutionJMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredSpectrophotometricallyatawavelengthof450nm.ComparedwiththeCUTOFFvalue,accordingtothistojudgeTAPPexistinthesampleornot.MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8CNegativecontrol0.5ml×1bottle0.5ml×1bottle2-8Positivecontrol0.5ml×1bottle0.5ml×1bottle2-8HRP-Conjugatereagent3ml×1bottle6ml×1bottle2-8CSamplediluent3ml×1bottle6ml×1bottle2-8ChromogenSolutionA3ml×1bottle6ml×1bottle2-8ChromogenSolutionB3ml×1bottle6ml×1bottle2-8CStopSolution3ml×1bottle6ml×1bottle2-8washsolution(20ml×20fold)Xibottle(20ml×30f0d)×Ibottle2-8°CSpecimenrequirements1. serum-coagulationatroomtemperature10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.2. plasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.3. Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.4. cellculturesupernatant-detectsecretorycomponents,collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPBS(PH7.2-7.4),Cellconcentrationreached1million/ml,repeatedfreeze-thawcycles,damagecellsandreleaseofintracellularcomponents,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.5. Tissuesamples-Aftercuttingsamples,checktheweight,addPBS(PH7.2-7.4),Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8oCaftermelting,addPBS(PH7.4),HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant.6. extractassoonaspossibleafterSpecimencollection,andacrdingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan't,specimencanbekeptin-20topreserve,Avoidrepeatedfreeze-thawcycles.7. Can,tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure1 .Number:tosamplecorrespondmicrotitrationwellandNumberSequence,eachplateshouldbesetfemininecomparison2wells,masculinecomparison2wells,blankcomparison1well(don,taddsampleandHRP-Conjugatereagenttoblankcomparisonwell,othereachsteptheoperationaresame).2 .addsample:separatelyaddPositivecontrolandNegativecontrol50ltothePositiveandNegativewell.addSampledilution40ltotestingsamplewell,thenaddtestingsample10l.addsampletothebottomofE1.ISAplatescoatedwell,don,ttouchthewellwallasfaraspossible,andGentlymix.3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37。4 .Configurateliquid:30-fold(or20-fold)washsolutiondiluteduntil600ml,andreserve.5 .washing：UncoverClosureplatemembrane,discard1.iquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.6 .addenzyme:AddHRP-Conjugatereagent50ltoeachwell,excepttheblankwell.1.1 ncubate:Operationwith3.8 .washing:Operationwith5.9 .color：AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat3710 .Stopthereaction:AddStopSolution50ltoeachwell,Stopthereaction(thebluecolorchangetoyellowlor).11 .assay：takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.DeterminetheresultTestvalidity:theaverageofPositivecontrolwell1.00;theaverageofNegativecontrolwell0.15.CalculateCritical(CUTOFF):Critical=theaverageofNegativecontrolwell+0.10.Negativecontrol:sampleOD<CalculateCriticaI(Cl)TOFF)isTAPPNegativecontrol.Positivecontrol:ampleODCalculateCritical(CUTOFF)isTAPPPositivecontrol.Importantnotes12 Pleaseaccordingtouseinstructionstrictly,Donotmixreagentswiththosefromotherlots.13 Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperaturethenuse,E1.ISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.14 washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.15 Closureplatemembraneonlylimitsthedisposableuse,inordertoavoidtheoverlappingpollution16 Thesubstratepleaseevadethelightpreservation.17 Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard,whenusedual-wavelengthtoassay,Referencewavelengthis630nm.18 AIIsamples,washingbufferandeachkindofrejectshouldaccordingtoinfectivematerialprocess.StoppSolutionis2Msulphuricacid.Youmustpayattentiontosafewhenuse.Storageandvalidity1. Storage:2-8.2. validity:sixmonths.