猴风疹病毒抗体IgMRVIgM酶联免疫分析ELISA试剂盒利用说明书

1、猴风疹病毒抗体IgM（RV-IgM）酶联免疫分析（ELISA）试剂盒利用说明书本试剂仅供研究利用目的：本试剂盒用于检测猴血清，血浆中风疹病毒抗体IgM（RV-IgM）水平，感染人的血液学诊断。实验原理：本试剂盒采用双抗原夹心酶联免疫法（ELISA）测定标本中猴风疹病毒抗体IgM（RV-IgM）。用纯化的抗原包被微孔板，制成固相抗原，可与样品中风疹病毒抗体IgM（RV-IgM）相结合，经洗涤除去未结合的抗体和其他成份后再与HRP标记的抗原结合，形成抗原T亢体-酶标抗原复合物，通过完全洗涤后加底物TMB显色。TMB住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。用酶标仪在450nm波

2、长下测定吸光度（0D值），与CUTOFF值相较较，从而判定标本中猴风疹病毒抗体Ig'KRV-IgM）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1X481X962-8C保存阴性对照XI瓶XI瓶2-8C保存阳性对照XI瓶XI瓶2-8C保存酶标试剂3mlXl瓶6mlXl瓶2-8C保存样品稀释液3mlXl瓶6mlX1瓶2-8保存显色剂A液3mlX1瓶6mlX1瓶2-8C保存显色剂B液3mlXl瓶6mlX1瓶2-8保存终止液3mlX1瓶6mlX1瓶2-8C保存浓缩洗涤液(20mlX20倍)XI瓶(20mlX30倍

3、)XI瓶2-8保存样本处置及要求：1 .血清：室温血液自然凝固10-20分钟，离心20分钟左右（2OOO-3OOO转/分）。认真搜集上清，保留进程中如显现沉淀，应再次离心。2 .血浆：应依照标本的要求选择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（2OOO-3OOO转/分）。认真搜集上清，保留进程中如有沉淀形成，应该再次离心。3 .尿液：用无菌管搜集，离心20分钟左右（2000-3000转/分）。认真搜集上清，保留进程中如有沉淀形成，应再次离心。胸腹水、脑脊液参如实行。4 .细胞培育上清：检测分泌性的成份时，用无菌管搜集。离心20分钟左右（2000-3000转/分）。

4、认真搜集上清。检测细胞内的成份时，用PBS（）稀释细胞悬液，细胞浓度达到100万/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）。认真搜集上清。保留进程中如有沉淀形成，应再次离心。5 .组织标本：切割标本后，称取重量。加入必然量的PBS,。用液氮迅速冷冻保留备用。标本融化后仍然维持2-8的温度。加入必然量的PBS（）,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）。认真搜集上清。分装后一份待检测，其余冷冻备用。6 .标本搜集后及早进行提取，提取按相关文献进行，提取后应尽快进行实验。假设不能马上进行实验，可将标本放于

5、-20保留，但应幸免反复冻融.7 .不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（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酶标试剂，其余各步操作相同）2 .加样：别离在阴、阳性对照孔中加入阴性对照、阳性对照50诅.然后在待测样品孔先加样品稀释液40山，然后再加待测样品10可。加样将样品加于酶标板孔底部，尽可能不触及孔壁，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：警惕揭掉封板膜，弃去液体，甩干

6、，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50m，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A50可，再加入显色剂B50pl,轻轻震荡混匀，37避光显色15分钟10 .终止：每孔加终止液50皿，终止反映（现在蓝色立转黄色）。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分钟之内进行。结果判定：实验有效性：阳性对照孔平均值三阴性对照平均值W临界值（CUTOFF）计算：临界值=阴性对照孔平均值十阴性判定：样品OD值临界值（CUTOFF）者为风疹病毒IgM（RV-Ig

7、M）抗体阴性阳性判定：样品OD值N临界值（CUTOFF）者为风疹病毒IgM（RV-IgM）抗体阳性注意事项1 .操作严格依照说明书进行，本试剂不同批号组分不得混用。2 .试剂盒从冷藏环境中掏出应在室温平稳15-30分钟后方可利用，酶标包被板开封后如未用完，板条应装入密封袋中保留。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，洗涤时不阻碍结果。4 .封板膜只限一次性利用，以幸免交叉污染。5 .底物请避光保留。6 .实验结果判定必需以醯标仪读数为准，利用双波长检测时，参考波长为630nm7 .所有样品，洗涤液和各类废弃物都应按传染物处置。终止液为2M的硫酸，利历时必需注意平安。保留条件

8、及有效期1 .试剂盒保留:2-832 .有效期：6个月RDHumanRV-IgMantibodyFORRESEARCHUSEONLYDrugNamesGenericName:HumanRV-IgMAbELISAKit.PurposeThiskitallowsforthedeterminationofRV-IgMAbconcentrationsinHumanserum,andotherbiologicalfluids.PrincipleoftheassayThekitassayRV-IgMAblevelinthesample,usePurifiedantigentocoatmicrotiterp

9、latewells,makesolid-phaseantigen,thenaddRV-IgMantibodytowells,CombinedWithantigen,afterwashingandremovingnon-combinativeantigenandothercomponents,thenCombinedantigenwhichwithHRPlabeledbecomeantigen-antibody-enzyme-antigencomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluec

10、olorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.ComparedwiththeCUTOFFvalue,accordingtothistojudgeRV-IgMAbexistinthesampleornot.MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinatio

11、ns96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8NegativecontrolX1bottleX1bottle2-8PositivecontrolX1bottleX1bottle2-8HRP-Conjugatereagent3mlx1bottle6mlx1bottle2-8Samplediluent3mlx1bottle6mlx1bottle2-8ChromogenSolutionA3mlx1bottle6mlx1bottle2-8ChromogenS

12、olutionB3mlx1bottle6mlx1bottle2-8StopSolution3mlx1bottle6mlx1bottle2-8washsolution(20mlX20fold)x1bottle(20mlX30fold)xIbottle2-8Specimenrequirements1. serum-coagulationatroomtemperature10-20mins,centrifugation20-minatthespeedof2000-3000removesupernatant,Ifprecipitationappeared,Centrifugalagain.2. pla

13、sma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000removesupernatant,Ifprecipitationappeared,Centrifugalagain.3. Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000removesupernatant,Ifprecipitationappeared,Centrifugalagain.

14、TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.4. cellculturesupernatant-detectsecretorycomponents,collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000removesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPBS(),Cellconcentrationreached1millionIml,repeated

15、freeze-thawcycles,damagecellsandreleaseofintracellularcomponents,centrifugation20-minatthespeedof2000-3000removesupernatant,Ifprecipitationappeared,Centrifugalagain.5. Tissuesamples-Aftercuttingsamples,checktheweight,addPBS(),Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8aftermelting,addPBS(),

16、HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000removesupernatant.6. extractassoonaspossibleafterSpecimencollection.andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan*t,specimencanbekeptin-20七topreserve,Avoidrepeatedfreeze-thawcyc

17、les.7. Can'tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure:tosamplecorrespondmicrotitrationwellandNumberSequence,eachplateshouldbesetfemininecomparison2wells,masculinecomparison2wells,blankcomparison1well(donJtaddsampleandHRP-Conjugatereagenttoblankcomparisonwell,oth

18、ereachsteptheoperationaresame).sample:separatelyaddPositivecontrolandNegativecontrol50pltothePositiveandNegativewell.addSampledilution40pltotestingsamplewell,thenaddtestingsample10pl.addsampletothebottomofELISAplatescoatedwell,don*ttouchthewellwallasfaraspossible,andGentlymix.:AfterclosingplatewithC

19、losureplatemembrane,incubatefor30minat37°C.liquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwateruntil600ml,andreserve.:UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.enzyme:AddHRP-Conjugatere

20、agent50pltoeachwell,excepttheblankwell.:Operationwith3.:Operationwith5.:AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37thereaction:AddStopSolution50pltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).11.assay：takeblankwellaszero,Readabsorbance

21、at450nmafterAddingStopSolutionandwithin15min.DeterminetheresultTestvalidity:theaverageofPositivecontrolwell;theaverageofNegativecontrolwellWCalculateCritical(CUTOFF):Critical"theaverageofNegativecontrolwell+.Negativecontrol:sampleOD<CalculateCritical(CUTOFF)isRV-IgMAbNegativecontrol.Positivecontrol:ampleOD'CalculateCritical(CUTOFF)isRV-IgMAbPositivecontrol.Importantnotesaccordingtouseinstructionstrictly,Donotmixreagentswiththosefromotherlots.kittakesoutfromt