人柯薩奇病(CoxV)酶聯(lián)免疫分析(ELISA)

#/6人柯薩奇?。?）酶聯(lián)免疫分析（ ）試劑盒使用說明書本試劑僅供研究使用目的：本試劑盒用于檢測人血清，血漿及相關液體樣本中柯薩奇?。–oxV）水平。目..潤屬釤瘞歌櫪廡賴。實驗原理：本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA）測定標本中人柯薩奇?。–oxV）。用純化的人柯薩奇?。–oxV）抗體包被微孔板，制成固相抗體，可與樣品中柯薩奇?。–oxV）相結合，經(jīng)洗滌除去未結合的抗體和其他成分后再與HRP標記的柯薩奇?。–oxV）抗體結合，形成抗體-抗原-酶標抗體復合物，經(jīng)過徹底洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成最終的黃色。用酶標儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標本中人柯薩奇?。–oxV）的存在與否。置試劑盒組成：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1個酶標包被板1x481x962-8℃保存陰性對照0.5mlx1瓶0.5mlx1瓶2-8℃保存陽性對照0.5mlx1瓶0.5mlx1瓶2-8℃保存酶標試劑3mlx1瓶6mlx1瓶2-8℃保存樣品稀釋液3mlx1瓶6mlx1瓶2-8℃保存顯色劑A液3mlx1瓶6mlx1瓶2-8℃保存顯色劑B液3mlx1瓶6mlx1瓶2-8℃保存終止液3mlx1瓶6mlx1瓶2-8℃保存濃縮洗滌液(20mlx20倍)x1瓶(20mlx30倍)x1瓶2-8℃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/分）。仔細收集上清，保存過程中如出現(xiàn)沉淀，應再次離心。殘騖樓.錈瀚湃溆塑箱。HHIIHIIIIII血漿：應根據(jù)標本的要求選擇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/分）。仔細收集上清，保存過程中如有沉淀形成，應該再次離心。釅錒極額朗鎮(zhèn)檜豬［夬錐。尿液：用無菌管收集，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。弓例攝爾霽斃撰磚鹵廡。IHI細胞培養(yǎng)上清：檢測分泌性的成份時，用無菌管收集。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。檢測細胞內(nèi)的成份時，用PBS（PH7.2-7.4）稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內(nèi)成份。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。W蕎摶篋一懟類蔣氤||||||||||||||||||||||||||||||||||||||||||||組織標本：切割標本后，稱取重量。加入一定量的PBS,PH7.4。用液氮迅速冷凍保存?zhèn)溆?。標本融化后仍然保?-8℃的溫度。加入一定量的PBS（PH7.4）,用手工或勻漿器將標本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。廈礴懇蹣駢―繼一■mmiiiiiiiiinii.標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20℃保存，但應避免反復凍融.煢楨廣鰳―選塊網(wǎng)踴淚。1.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：1.編號：將樣品對應微孔按序編號，每板應設陰性對照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試劑，其余各步操作相同）鵝婭盡損鵪慘屣蘢一。BH加樣：分別在陰、陽性對照孔中加入陰性對照、陽性對照50m。然后在待測樣品孔先加樣品稀釋液4om，然后再加待測樣品iom。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻，箱叢媽.為贍僨蟶練浮。.溫育：用封板膜封板后置37℃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5次，拍干。.加酶：每孔加入酶標試劑50m，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A50m，再加入顯色劑B50m，輕輕震蕩混勻，37℃避光顯色15分鐘.終止：每孔加終止液50亂終止反應（此時藍色立轉黃色）。測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。測定應在加終止液后15分鐘以內(nèi)進行。——艮訝驊糴。結果判定：試驗有效性：陽性對照孔平均值21.00；陰性對照平均值戌.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）者為人柯薩奇?。–oxV）陰性陽性判定：樣品OD值2臨界值（CUTOFF）者為人柯薩奇病（CoxV）陽性注意事項1．操作嚴格按照說明書進行，本試劑不同批號組分不得混用。2．試劑盒從冷藏環(huán)境中取出應在室溫平衡15-30分鐘后方可使用，酶標包被板開封后如未用完，板條應裝入密封袋中保存。滲彭嗆儼勻—帛。3．濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。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。.試驗結果判定必須以酶標儀讀數(shù)為準，使用雙波長檢測時，參考波長為630nm.所有樣品，洗滌液和各種廢棄物都應按傳染物處理。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1．試劑盒保存：；2-8℃。2．有效期：6個月RRBHumanCoxsakievirus(CoxV)FORRESEARCHUSEONLYDrugNamesGenericName：HumanCoxsakievirus(CoxV)ELISAKit.PurposeThiskitallowsforthedeterminationofCoxVconcentrationsinHumanserum,andotherbiologicalfluids.鐃^臥瀉.圣騁貺丁直廉。PrincipleoftheassayThekitassayCoxVlevelinthesample，usePurifiedCoxVantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddCoxVtowells,CombinedWithCoxV,afterwashingandremovingnon-combinativeantibodyandothercomponents,thenCombinedCoxVantibodywhichwithHRPlabeledbecomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.ComparedwiththeCUTOFFvalue,accordingtothistojudgeCoxVexistinthesampleornot.挑冬帝鳳襪備^^輪爛薔。MaterialsprovidedwiththekitMaterialsprovidedwiththekit48determinations96determinationsStorageUsermanual11Closureplatemembrane22Sealedbags11Microelisastripplate112-8℃Negativecontrol0.5mlx1bottle0.5mlx1bottle2-8℃Positivecontrol0.5mlx1bottle0.5mlx1bottle2-8℃HRP-Conjugatereagent3mlx1bottle6mlx1bottle2-8℃Samplediluent3mlx1bottle6mlx1bottle2-8℃ChromogenSolutionA3mlx1bottle6mlx1bottle2-8℃ChromogenSolutionB3mlx1bottle6mlx1bottle2-8℃StopSolution3mlx1bottle6mlx1bottle2-8℃washsolution(20mlx20fold)xlbottle(20mlx30fold)x1bottle2-8℃Specimenrequirements1.serum-coagulationatroomtemperature10-20mins，centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalaga0熟俁閫羲Uplasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.壇搏鄉(xiāng)舅懺蔞鍥鈴覲波。Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.蠟燮修.輟倀鉉錨—贅。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.^鯛—曇庸遙閆擷凄。Tissuesamples-Aftercuttingsamples,checktheweight,addPBS(PH7.2-7.4),Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8℃aftermelting,addPBS(PH7.4),HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant.麥鏑蜩^^鶘蹤韋轔汆翟。extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.,^躓震彥浹綏言.飴夏錦。Can'tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.貓蠆^繪燎蒯誅髏既廡。Assayprocedure.Number:tosamplecorrespondmicrotitrationwellandNumberSequence,eachplateshouldbesetfemininecomparison2wells,masculinecomparison2wells,blankcomparison1well(don’taddsampleandHRP-Conjugatereagenttoblankcomparisonwell,othereachsteptheoperationaresame).鍬籟饕逕瑣肇禊鷗婭薔。.addsample：separatelyaddPositivecontrolandNegativecontrol50MtothePositiveandNegativewell.addSampledilution40Mtotestingsamplewell,thenaddtestingsample10M.addsampletothebottomofELISAplatescoatedwell,don’ttouchthewellwallasfaraspossible,andGentlymix.橫氽元直黌碩飩薺齦話騖。.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37℃.輒嶧隨4.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwateruntil600ml,andreserve.葬側閨藕絳^絢勵.瞽。.washing：UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.識鐮諼金昆縊激竟.儼凄。.addenzyme：AddHRP-Conjugatereagent50gltoeachwell,excepttheblankwell.沫—勞月葭鍇癇嫦脛糴。7.incubate：Operationwith3.8.washing：Operationwith5.9.color：AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37℃耳心—滅縈歡蜴聾鶩金帛。10.Stopthereaction：AddStopSolution50mtoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).鯊胃輸詘襁御溈，?瞿統(tǒng)康。11.assay：takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.碩痍鄭頏謅撞棒摧,—蘞。DeterminetheresultTestvalidity:theaverageofPositivecontrolwell>1.00;theaverageofNegativecontrolwell閿擻一^^遷擇一秘麓。CalculateCritical(CUTOFF):Critical=theaverageofNegativecontrolwell+0.15.氮?！毁Q(mào)懇彈濾頷果。Negativecontrol:sampleOD<CalculateCritical(CUTOFF)isCoxVNegativecontrol.金土鵒資贏?^孫7題肺贅。Positivecontrol:ampleOD>CalculateCritical(CUTOFF)isCoxVPositivecontrol.慫闡^.逕醇嘯重房涼。Importantnotes.Pleaseaccordingtouseinstructionstrictly,Donotmixreagentswiththosefromother10ts.諺辭^擔金亢諂動律瀉類一.Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesin