人髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG

人髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)酶聯(lián)免疫分析試劑盒使用說明書本試劑盒僅供研究使用。96T使用目的：本試劑盒用于測定人血清、血漿及相關(guān)液體樣本中髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)表達(dá)。實(shí)驗(yàn)原理本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中人髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)表達(dá)。用純化的抗體包被微孔板，制成固相抗體，可與樣品中髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)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)記的抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(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)本中人髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)的存在與否。試劑盒組成130倍濃縮洗滌液20mlX1瓶7終止液6mlX1瓶2酶標(biāo)試劑6mlX1瓶8陽性對照0.5mlX1瓶3酶標(biāo)包被板12孔X8條9陰性對照0.5mlX1瓶4樣品稀釋液6mlX1瓶10說明書1份5顯色劑A液6mlX1瓶11封板膜2張6顯色劑B液6mlX1/瓶12密封袋1個標(biāo)本要求1．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：將樣品對應(yīng)微孔按序編號，每板應(yīng)設(shè)陰性對照2孔、陽性對照2孔、空白對照1孔(空白對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、陽性對照孔中加入陰性對照、陽性對照50卩1。然后在待測樣品孔先加樣品稀釋液40卩1，然后再加待測樣品10卩1。加樣將樣品加于酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動混勻，溫育：用封板膜封板后置37C溫育30分鐘。配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用洗滌：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50卩1，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A50yl，再加入顯色劑B50yl，輕輕震蕩混勻，37°C避光顯色15分鐘.終止：每孔加終止液50卩1，終止反應(yīng)（此時藍(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。操作程序總結(jié)：計算和結(jié)果判定：試驗(yàn)有效性：陽性對照孔平均臉1.00;陰性對照平均值三0.10臨界值（CUTOFF）計算：臨界值=陰性對照孔平均值+0.15陰性判定：樣品OD值＜臨界值（CUTOFF）者為髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)陰性陽性判定：樣品OD值〉臨界值(CUTOFF)者為髓過氧化物酶特異性抗中性粒細(xì)胞胞質(zhì)抗體IgG(MPO-ANCAIgG)陽性。注意事項(xiàng)1．操作嚴(yán)格按照說明書進(jìn)行，本試劑不同批號組分不得混用。2．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。3．濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。4．封板膜只限一次性使用，以避免交叉污染。5．底物請避光保存。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，參考波長為630nm7?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個月RDHumanMPO-ANCAIgGFORRESEARCHUSEONLY96determinationsPurposeThiskitallowsforthedeterminationofMPO-ANCAIgGconcentrationsinHumanserum,andotherbiologicalfluids.PrincipleoftheassayThekitassayMPO-ANCAIgGlevelinthesample，usePurifiedantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddMPO-ANCAIgGtowells,CombinedWithMPO-ANCAIgG,afterwashingandremovingnon-combinativeantibodyandothercomponents,thenCombinedantibodywhichwithHRPlabeledbecomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.ComparedwiththeCUTOFFvalue,accordingtothistojudgeMPO-ANCAIgGexistinthesampleornot.Materialsprovidedwiththekit1washsolution20mlx1bottle7StoppSolution6mlx1bottle2HRP-Conjugatereagent6mlx1bottle8Positivecontrol0.5mlx1bottle3Microelisastripplate12wellx8strips9Negativecontrol0.5mlx1bottle4Samplediluent6mlx1bottle10Instruction15ChromogenSolutionA6mlx1bottle11Closureplatemembrane26ChromogenSolutionB6mlx1bottle12Sealedbags1SpecimenrequirementsextractassoonaspossibleafterSpecimencollection,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=theaverageofNegativecontrolwell+0.15.Negativecontrol:sampleOD<CalculateCritical(CUTOFF)isMPO-ANCAIgGNegativecontrol.Positivecontrol:ampleOD>CalculateCritical(CUTOFF)isMPO-ANCAIgGPositivecontrol.ImportantnotesPleaseaccordingtouseinstructionstrictly,Donotmixreagentswiththosefromotherlots.Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperaturethenuse,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.washingbufferwillCrystallizationseparation,itcanbeheated