鸡γ干扰素IFNγ检测试剂盒.docx

ChickenIFN-FORRESEARCHUSEON1YAssayrange：1.5ng1-48ng196determinationsPurposeThiskita11owsforthedeterminationofIFN-concentrationsinChickenserum,ce11cu1tureSupernatesandotherbio1ogica1f1uidsPrincip1eoftheassayThekitassayChickenIFN-1eve1inthesamp1e,usePurifiedChickenIFN-antibodytocoatmicrotiterp1atewe11s,makeso1id-phaseantibody,thenaddIFN-towe11s,CombinedIFN-antibodywhichWithHRP1abe1ed,bemeantibody-antigen-enzyme-antibodycomp1ex,afterwashingComp1ete1y,AddTMBsubstrateSoIution1TMBsubstratebecomesb1ueco1orAtHRPenzyme-cata1yzed,reactionisterminatedbytheadditionofasu1phuricacidso1utionandthe1orchangeismeasuredSpectrophotometrica11yatawave1engthof450nm.TheconcentrationofChickenIFN-inthesamp1esisthendeterminedbycomparingthe0.D.ofthesamp1estothestandardcurve.Materia1sprovidedwiththekit1washso1ution20m1×1bott1e7StopSo1utionm1×1bott1e2HRP-Conjugatereagent6m1×1bott1e8Standard(96ng1)0.5m1×1bott1e3Microe1isastripp1ate12we11×8strips9Standarddi1uent1.5m1×1bott1e4Samp1edi1uent6m1×1bott1e10Instruction15ChromogenSo1utionA6m1×1bott1e11C1osurep1atemembrane26ChromogenSo1utionB6m1×1bott1e12Sea1edbags1Specimenrequirements1. extractassoonaspossib1eafterSpecimenco11ection,andaccordingtothere1evant1iterature,andshou1dbeexperimentassoonaspossib1eaftertheextraction.Ifitcan't,specimencanbekeptin-20topreserve,Avoidrepeatedfreeze-thawcyc1es.2. Can'tdetectthesamp1ewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure1.Di1uteandaddsamp1e:Di1uteOrigina1densityStandardasfo11owtab1e:48ng15Standard1501Origina1densityStandard+1501Standarddi1uent24ng14Standard15015Standard+1501Standarddi1uent12ng13Standard15014Standard+1501Standarddi1uent6ng12Standard15013Standard+1501Standarddi1uent3ng11Standard15012Standard+1501Standarddi1uent3. addsamp1e：Setb1ankwe11sseparate1y(b1ankcomparisonwe11sdon'taddsamp1eandHRP-Conjugatereagent,othereachstepoperationissame),testingsamp1ewe11,addSamp1edi1ution401totestingsamp1ewe11,thenaddtestingsamp1e101(samp1efina1di1utionis5-fo1d),addsamp1etowe11s,don'ttouchthewe11wa11asfaraspossib1e,andGent1ymix.3.Incubate:Afterc1osingp1atewithC1osurep1atemembrane,incubatefor30minat37.4.Configurate1iquid:30-fo1d(or20-fo1d)washso1utiondi1uted30-fo1d(or20-fo1d)withdisti11edwaterandreserve.5.washing：UncoverC1osurep1atemembrane,discard1iquid,drybyswing,addwashingbuffertoeverywe11,sti11for30sthendrain,repeat5times,drybypat.addenzyme：AddHRP-Conjugatereagent501toeachwe11,exceptb1ankwe11.7.incubate:Operationwith3.8.washing：Operationwith5.9.co1or:AddChromogenSo1utionA50u1andChromogenSo1utionBtoeachwe11,evadethe1ightpreservationfor10minat3710.Stopthereaction:AddStopSo1ution501toeachwe11,Stopthereaction(theb1ueco1orchangetoye11owco1or).11.assay:takeb1ankwe11aszero,Readabsorbanceat450nmafterAddingStopSo1utionandwithin15min.StepsdescriptionStandard,Samp1edi1uentAddStandard,Samp1edi1uent,incubatefor30minat37.Wash5time1AddHRP-Conjugatereagent,incubatefor30minat37.Wash5times,AddChromogenSoIutionAandB,incubatefor10minat37.AddStopSo1utionCa1cu1ateTakethestandarddensityasthehorizonta1,theODva1ueforthevertica1,drawthestandardcurveongraphpaper,Findoutthecorrespondingdensityaccordingtothesamp1eODva1uebytheSamp1ecurve,mu1tip1iedbythedi1utionmu1tip1e,orca1cu1atethestraight1ineregressionequationofthestandardcurvewiththestandarddensityandtheODva1ue,withthesamp1eODva1ueintheequation,ca1cu1atethesamp1edensity,mu1tip1iedbythedi1utionfactor,theresu1tisthesamp1eactua1density.Importantnotes1. Thekittakesoutfromtherefrigerationenvironmentshou1dbeba1anced15-30minutesintheroomtemperature,E1ISAp1atescoatedifhasnotuseupafteropened,thep1ateshou1dbestoredinSea1edbag.2. washingbufferwi11Crysta11izationseparation,itcanbeheatedthewaterhe1psdisso1vewhendi1ute.Washingdoesnotaffecttheresu1t.3. addSamp1ewithsamp1erEachstep,Andproofreaditsaccuracyfrequent1y,avoidstheexperimenta1error,addsamp1ewithin5min,ifthenumberofsamp1eismuch,recommendtouseVo11ey.4. ifthetestingmateria1contentisexcessive1yhigher(Thesamp1eODisbiggerthanthefirststandardwe11),p1easedi1uteSamp1e(n-fo1d),P1easedi1uenteandmu1tip1iedbythedi1utionfactor,(×n×5).5. C1osurep1atemembraneon1y1imitsthedisposab1euse,toavoidcross-contamination.6. Thesubstrateevadethe1ightpreservation.7. P1easeaccordingtouseinstructionstrict1y,Thetestresu1tdeterminationmusttakethemicrotiterp1atereaderasastandard.8. A11samp1es,washingbufferandeachkindofrejectshou1daccordingtoinfectivemateria1process.9. Donotmixreagentswiththosefromother1ots.Storageandva1idity1. Storage:2-8.2. va1idity:sixmonths