人粒细胞集落刺激因子(G-CSF)酶联免疫分析试剂盒使用说明书

人粒细胞集落刺激因子〔G-CSF〕酶联免疫分析试剂盒使用说明书本试剂仅供争论使用目的：本试剂盒用于测定大鼠血清，血浆及相关液体样本中粒细胞集落刺激因子〔G-CSF 〕的含量。试验原理：本试剂盒应用双抗体夹心法测定标本中人粒细胞集落刺激因子〔G-CSF水平。用纯化的人粒细胞集落刺激因子〔G-CSF 被微孔板，制成固相抗体，往包被单抗的微孔中依次参与粒细胞集落刺激因子〔G-CSF〕，再与HRP标记的粒细胞集落刺激因子〔G-CSF〕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。颜色的深浅和样品的粒细胞集落刺激因子〔G-CSF波长下测定吸光度〔OD值〕，通过标准曲线计算样品中人粒细胞集落刺激因子〔G-CSF 〕浓度。试剂盒组成：48孔配置96孔配置保存1份1份2片〔48〕2片〔96〕1个1个1×481×962-8℃保存标准品：45ng/L0.5ml×10.5ml×12-8℃保存标准品稀释液1.5ml×1瓶1.5ml×1瓶2-8℃保存3ml×1瓶6ml×1瓶2-8℃保存3ml×1瓶6ml×1瓶2-8℃保存2-8℃保存2-8℃保存3ml×1瓶6ml×1瓶2-8℃保存〔20ml×20〕×1〔20ml×30〕×12-8℃保存样本处理及要求：20分钟左右〔2023-3000转/分〕。认真收集上清，保存过程中如消灭沉淀，应再次离心。10-20分钟后，离心20〔2023-3000/分〕。认真收集上清，保存过程中如有沉淀形成，应当再次离心。转/分〕。认真收集上清，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。细胞培育上清：检测分泌性的成份时，用无菌管收集。离心20分钟左右〔2023-3000/PBS〔PH7.2-7.4〕稀释细胞悬液，细胞220分钟左右〔2023-3000/分〕。认真收集上清。保存过程中如有沉淀形成，应再次离心。组织标本：切割标本后，称取重量。参与确定量的PBS，PH7.4。用液氮快速冷冻保存备用。标本溶化后照旧保持2-8℃的温度。参与确定量的PBS〔PH7.4〕，用手工或匀浆器〔2023-3000转/分〕。认真收集上清。分装后一份待检测，其余冷冻备用。假设不能马上进展试验，可将标本放于-20℃保存，但应避开反复冻融.抑制辣根过氧化物酶的〔HRP〕活性。操作步骤：10孔，在第一、其次孔中分别加标100μl，然后在第一、其次孔中加标准品稀释液50μl，混匀；然后从第一孔、其次50μl，50μl50μl分别加到第五、第六孔50ul，混匀；混匀后从第五、第六孔中各50μl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50μl，混加到第九、第十孔中，再在第九第十孔分别加标准50μl，30ng/L，20ng/L，10ng/L，5ng/L，2.5ng/L〕。〔、待测样40μl，然后再加待测样品10μl〔样倍轻晃动混匀。温育：用封板膜封板后置37℃温育30分钟。30〔48T20倍〕倍稀释后备用。30秒后弃5次，拍干。加酶：每孔参与酶标试剂50μl，空白孔除外。温育：操作同3。洗涤：操作同5。显色：每孔先参与显色剂A50μl，再参与显色剂B50μl，轻轻荡混匀，37℃避光显色15终止：每孔加终止液50μl，终止反响〔此时蓝色立转黄色〕。15分钟以内进展。留意事项：15-30板开封后如未用完，板条应装入密封袋中保存。果。样时间最好把握在5分钟内，如标本数量多，推举使用排枪加样。〔样OD3OD值倍〕后再测定，计算时请最终乘以总稀释倍数〔×n×5〕。封板膜只限一次性使用，以避开穿插污染。底物请避光保存。严格依据说明书的操作进展，试验结果判定必需以酶标仪读数为准.全部样品，洗涤液和各种废弃物都应按传染物处理。本试剂不同批号组分不得混用。计算：以标准物的浓度为横坐标，OD在坐标纸上绘出标准曲线，依据样品的OD值由标准曲线查出相应的浓度；再乘以稀释OD值计算出标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。〕试剂盒性能：R值为0.990以上。批内与批见应分别小于9%和11%检测范围：2ng/L-40ng/L保存条件及有效期：试剂盒保存：；2-8℃。有效期：6835041457或mobilecall一五一二一零七二二五三Ratinterleukin1βFORRESEARCHUSEONLYDrugNamesGenericName：Ratinterleukin1β(G-CSF)ELISAKit.PurposeThiskitallowsforthedeterminationofG-CSF concentrationsinRatserum,bloodplasma,andotherbiologicalfluids.PrincipleoftheassayThekitassayRatG-CSF levelinthesample,usePurifiedRatG-CSFantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddG-CSFtowells,CombinedG-CSFantibodywhichWithHRPlabeled,becomeantibody-antigen-enzyme-antibodycomplex,afterAddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofG-CSF determinedbycomparingtheO.D.ofthesamplestothestandardcurve.5MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8℃Standard：45ng/L0.5ml×1bottle0.5ml×1bottle2-8℃Standarddiluent1.5ml×1bottle1.5ml×1bottle2-8℃HRP-Conjugatereagent3ml×1bottle6ml×1bottle2-8℃Samplediluent3ml×1bottle6ml×1bottle2-8℃ChromogenSolutionA3ml×1bottle6ml×1bottle2-8℃ChromogenSolutionB3ml×1bottle6ml×1bottle2-8℃StopSolution3ml×1bottle6ml×1bottle2-8℃washsolution〔20ml×20fold〕×1bottle〔20ml×30fold〕×1bottle2-8℃Specimenrequirementsserum-coagulationatroomtemperature10-20mins，centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.EDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.cellculturesupernatant-detectsecretorycomponents,collectsueasterilecontainer,centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPSPH7.2-7.4〕,concentrationreached1million/ml,repeatedfreeze-thawcycles,damagecellsandreleaseofintracellularcomponents,centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.Tissuesamples-Aftercuttingsamples,checktheweight,addPBS〔PH7.2-7.4〕,Rapidlynitrogen,maintainsamplesat2-8℃aftermelting,addPBS〔PH7.4〕,6HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2023-3000r.p.m.removesupernatant.extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,in-20℃topreserve,Avoidrepeatedfreeze-thawcycles.samplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.AssayprocedureDiluteandaddsampletoStandard:set10StandardwellsontheELISAplatescoated,addStandard100μltothefirstandthesecondwell,thenaddStandarddilution50μltothefirstandthesecondwell,mix;takeoutthenaddittothethird

lformthefirstandthesecondwellandtheforthwellseparately.thenaddStandarddilution50μltothethirdandtheforthwell,mix;thentakeout50μlfromthethirdandtheforthwelldiscard,add50μltothefifthandthesixthwell,thenaddStandarddilution50μltothefifthandthesixthwell,mix;takeout50μlfromthefifthandthesixthwellandaddtotheseventhandtheeighthwell,thenaddStandarddilution50μltotheseventhandtheeighthwell,mix;takeout50μlfromtheseventhandtheaddtotheninthandthetenthwell,addStandarddilution50μltotheninthandthetenthwell,mix,takeout50μlfromtheninthandthetenthwelldiscard(addSample50μltoeachwellafterDiluting,(density:30ng/L,20ng/L,10ng/L,5ng/L,2.5ng/L)addsample：Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandothereachstepoperationissame).testingsamplewell.addSampledilution40μltotestingsamplewell,thenaddtestingsample10μl(samplefinaldilutionisandGentlymix.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37℃.30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwaterandreserve.Closureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.reagent50μltoeachwell,exceptblankwell.incubate：Operationwith3.7washing：Operationwith5.color：AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37℃Stopthereaction：AddStopSolution50μltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).blankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.importantnotesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesinELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheaddsamplewithin5mins,ifthenumberofsampleismuch,recommendtouseVolley.ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.〔×n×5〕.Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.Thesubstrateevadethelightpreservation.Pleaseaccordingtouseinstructionstrictly,Thetest