牛布氏桿菌抗體說明書定性

牛布氏桿菌抗體（Brucella-Ab）酶聯(lián)免疫分析（ELISA）試劑盒使用說明書本試劑僅供研究使用目的：本試劑盒用于檢測牛血清，血漿中布氏桿菌擠體（Brucella-Ab）水平。實(shí)驗(yàn)原理：本試劑盒采用雙抗原夾心酶聯(lián)免疫法（ELISA）測定標(biāo)本中牛布氏桿菌抗體（Brucella-Ab）0用純化的牛布氏桿菌抗原包被微孔板，制成固相抗原，可與樣品中布氏桿菌抗體（Brucella-Ab）相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗原和其他成分后再與HRP標(biāo)記的布氏桿菌抗原結(jié)合，形成抗原-抗體-酶標(biāo)抗原復(fù)合物，經(jīng)過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色。用酶標(biāo)儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標(biāo)本中牛布氏桿菌抗體（Brucella-Ab）的存在與否。試劑盒組成:試劑盒組成48孔配置96孔配置保存說明書1份1份封板膜2片(48)2片(96)密封袋1個(gè)1個(gè)酶標(biāo)包被板1X481X962-8°C保存陰性對(duì)照0.5mlX1瓶0.5mlX1瓶2-8°C保存陽性對(duì)照0.5mlX1瓶0.5mlX1瓶2-8C保存酶標(biāo)試劑3mlX1瓶6mlX1瓶2-8C保存樣品稀釋液3mlX1瓶6mlX1瓶2-8C保存顯色劑A液3mlX1瓶6mlX1瓶2-8C保存顯色劑B液3mlX1瓶6mlX1瓶2-8C保存終止液3mlX1瓶6mlX1瓶2-8C保存濃縮洗滌液(20mlX20倍)X1瓶(20mlX30倍)X1瓶2-8C保存樣本處理及要求：血清：室溫血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如出現(xiàn)沉淀，應(yīng)再次離心。血漿：應(yīng)根據(jù)標(biāo)本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)該再次離心。尿液：用無菌管收集，離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清，保存過程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。細(xì)胞培養(yǎng)上清：檢測分泌性的成份時(shí)，用無菌管收集。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。檢測細(xì)胞內(nèi)的成份時(shí)，用PBS（PH7.2-7.4）稀釋細(xì)胞懸液，細(xì)胞濃度達(dá)到100萬/ml左右。通過反復(fù)凍融，以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。保存過程中如有沉淀形成，應(yīng)再次離心。組織標(biāo)本：切割標(biāo)本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存?zhèn)溆?。?biāo)本融化后仍然保持2-8^的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右（2000-3000轉(zhuǎn)/分）。仔細(xì)收集上清。分裝后一份待檢測，其余冷凍備用。標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20°C保存，但應(yīng)避免反復(fù)凍融.不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。操作步驟：編號(hào)：將樣品對(duì)應(yīng)微孔按序編號(hào)，每板應(yīng)設(shè)陰性對(duì)照2孔、陽性對(duì)照2孔、空白對(duì)照1孑L（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）加樣：分別在陰、陽性對(duì)照孔中加入陰性對(duì)照、陽性對(duì)照50gl。然后在待測樣品孔先加樣品稀釋液40gl，然后再加待測樣品10gl。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動(dòng)混勻，溫育：用封板膜封板后置37C溫育30分鐘。配液：將30（48T的20倍）倍濃縮洗滌液加蒸餾水至600ml后備用洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復(fù)5次，拍干。加酶：每孔加入酶標(biāo)試劑50gl，空白孔除外。溫育：操作同3。洗滌：操作同5。顯色：每孔先加入顯色劑A50皿再加入顯色劑B50gl，輕輕震蕩混勻，37C避光顯色15分鐘終止：每孔加終止液50gl，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。測定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。結(jié)果判定：試驗(yàn)有效性：陽性對(duì)照孔平均隹1.00;陰性對(duì)照平均值＜0.10臨界值（CUTOFF）計(jì)算：臨界值=陰性對(duì)照孔平均值+0.15陰性判定：樣品OD值＜臨界值（CUTOFF）者為牛布氏桿菌抗體（Brucella-Ab）陰性陽性判定：樣品OD值2臨界值（CUTOFF）者為牛布氏桿菌抗體（Brucella-Ab）陽性注意事項(xiàng)操作嚴(yán)格按照說明書進(jìn)行，本試劑不同批號(hào)組分不得混用。試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘后方可使用，酶標(biāo)包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。封板膜只限一次性使用，以避免交叉污染。5.底物請(qǐng)避光保存。6.試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，使用雙波長檢測時(shí)，參考波長為630nm所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。終止液為2M的硫酸，使用時(shí)必須注意安全。保存條件及有效期試劑盒保存:；2-8°C。有效期：6個(gè)月RBBovineBrucella-AbFORRESEARCHUSEONLYDrugNamesGenericName:BovineBrucella-AbELISAKit.PurposeThiskitallowsforthedeterminationofBrucella-AbconcentrationsinBovineserum,andotherbiologicalfluids.PrincipleoftheassayThekitassayBrucella-Ablevelinthesample,usePurifiedBrucellaantigentocoatmicrotiterplatewells,makesolid-phaseantigen,thenaddBrucella-Abtowells,CombinedWithBrucella-Ab,afterwashingandremovingnon-combinativeantigenandothercomponents,thenCombinedBrucellawhichwithHRPlabeledbecomeantigen-antibody-enzyme-antigencomplex,afterwashingCompletely,AddTMBsubstratesolution,,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.ComparedwiththeCUTOFFvalue,accordingtothistojudgeBrucella-Abexistinthesampleornot.

MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8°CNegativecontrol0.5mlx1bottle0.5mlx1bottle2-8°CPositivecontrol0.5mlx1bottle0.5mlx1bottle2-8CHRP-Conjugatereagent3mlx1bottle6mlx1bottle2-8CSamplediluent3mlx1bottle6mlx1bottle2-8CChromogenSolutionA3mlx1bottle6mlx1bottle2-8CChromogenSolutionB3mlx1bottle6mlx1bottle2-8CStopSolution3mlx1bottle6mlx1bottle2-8Cwashsolution(20mlX20fold)xibottle(20mlX30fold)X1bottle2-8CSpecimenrequirementsserum-coagulationatroomtemperature10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.plasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.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.Tissuesamples-Aftercuttingsamples,checktheweight,addPBS(PH7.2-7.4),Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8°Caftermelting,addPBS(PH7.4),HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant.extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20°Ctopreserve,Avoidrepeatedfreeze-thawcycles.Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.AssayprocedureNumber:tosamplecorrespondmicrotitrationwellandNumberSequence,eachplateshouldbesetfemininecomparison2wells,masculinecomparison2wells,blankcomparison1well(don’taddsampleandHRP-Conjugatereagenttoblankcomparisonwell,othereachsteptheoperationaresame).addsample:separatelyaddPositivecontrolandNegativecontrol50pltothePositiveandNegativewell.addSampledilution40pltotestingsamplewell,thenaddtestingsample10pl.addsampletothebottomofELISAplatescoatedwell,don'ttouchthewellwallasfaraspossible,andGentlymix.3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37C.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwateruntil600ml,andreserve.washing:UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.addenzyme:AddHRP-Conjugatereagent50pltoeachwell,excepttheblankwell.7.incubate:Operationwith3.washing:Operationwith5.color:AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37C10.Stopthereaction:AddStopSolution50pltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).11.assay:takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.DeterminetheresultTestvalidity:theaverageofPositivecontrolwell>1.00;theaverageofNegativecontrolwell<0.10.CalculateCritical(CUTOFF):Critical二theaverageofNegativecontrolwell+0.15.Negativecontrol:sampleOD<CalculateCritical(CUTOFF)isBruceiia-AbNegativecontrol.Positivecontrol:ampleOD>CalculateCritical(CUTOFF)isBruceiia-AbPositivecontrol.ImportantnotesPleaseaccordingtouseinstructionstrictly,Donotmixreagentswiththosefromotherlots.Thekittakesoutfromtherefrigerationenvironments