大鼠P选择素(PSelectin)酶联免疫分析试剂盒英文使用说明书

RatP-Selectin
FORRESEARCHUSEONLY 
DrugNames
GenericName：RatP-Selectin(PS)ELISAKit.
Purpose
ThiskitallowsforthedeterminationofPSconcentrationsinRatserum,bloodplasma,andotherBIOLOGicalfluids.
Principleoftheassay
ThekitassayRatPSlevelinthesample，usePurifiedRatPStocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddPStowells,CombinedPSwhichWithHRPlabeled，becomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofPSinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.
 
 
 
 
 
 
 
MaterialsprovidedwiththekitSpecimenrequirements

1. serum-coagulationatroomtemperature10-20mins，centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.

2. plasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.

3. Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.

4. 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.

5. Tissuesamples-Aftercuttingsamples,checktheweight,addPBS（PH7.2-7.4）,Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8℃aftermelting,addPBS（PH7.4）,HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant.

6. extractassoonaspossIBLeafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.

7. Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.Assayprocedure
   1.DiluteandaddsampletoStandard:set10StandardwellsontheELISAplatescoated,addStandard100μltothefirstandthesecondwell,thenaddStandarddilution50μltothefirstandthesecondwell,mix;takeout100μlformthefirstandthesecondwellthenaddittothethirdandtheforthwellseparately.thenaddStandarddilution50μltothethirdandtheforthwell,mix;thentakeout50μlfromthethirdandtheforthwelldiscard,add50μltothefifthandthesixthwell,thenaddStandarddilution50μltothefifthandthesixthwell,mix;takeout50μlfromthefifthandthesixthwellandaddtotheseventhandtheeighthwell,thenaddStandarddilution50μltotheseventhandtheeighthwell,mix;takeout50μlfromtheseventhandtheeighthwellandaddtotheninthandthetenthwell,addStandarddilution50μltotheninthandthetenthwell,mix,takeout50μlfromtheninthandthetenthwelldiscard(addSample50μltoeachwellafterDiluting,(density:24ng/L,16ng/L,8ng/L,4ng/L,2ng/L)
   2.addsample：Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandHRP-Conjugatereagent,othereachstepoperationissame).testingsamplewell.addSampledilution40μltotestingsamplewell,thenaddtestingsample10μl(samplefinaldilutionis5-fold),addsampletowells,don’ttouchthewellwallasfaraspossible,andGentlymix.
   3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37℃.
   4.Configurateliquid:washsolutiondiluted20-foldwithdistilledwaterandreserve.
   5.washing：UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.
   6.addenzyme：AddHRP-Conjugatereagent50μltoeachwell,except blankwell.
   7.incubate：Operationwith3.
   8.washing：Operationwith5.
   9.color：AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37℃
   10.Stopthereaction：AddStopSolution50μltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).
   11.assay：takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.
   Importantnotes

1. Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.

2. washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.

3. addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5mins,ifthenumberofsampleismuch,recommendtouseVolley.

4. ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.（×n×5）.

5. Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.

6. Thesubstrateevadethelightpreservation.

7. Pleaseaccordingtouseinstructionstrictly,Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.

8. Allsamples,washingbufferandeachkindofrejectshouldaccordingtoinfectivematerialprocess.

9. Donotmixreagentswiththosefromotherlots. 
    
   Assayrange
   1ng/L-30ng/L
    
   Storageandvalidity
   1．Storage： 2-8℃.
   2．validity：sixmonths.