SANDWICH ELISA FOR DETECTING SECRETED ICAM1
来自 : 蚂蚁淘
Materials
- NUNC-ImmunoPlateIIF.
- HorserADIshPeroxidase-Streptavidin(Zymed;43-4323)
- PBSorBalancedSaltSolution(BSS).DonotuseRPMIoranymediumthatcontainsbiotin.
- BSA(1%inPBS).
- ABTSsubstrateandsubstratebuffer(Zymed;#00-2011)
- ABTSchromogensolution
- HydrogenPeroxidasesolution
- Citratebuffer
Anti-ICAM-1mAbR6.5orCL203.Useas10ug/mlinPBStocoatplates.BiotinylatedmAb:BiotinRR1/1,R6.1andBiotinR6.5supplied.- Useatafinalconcentrationof2ug/ml(1:1000dilution).
- Donotrefreeze.Stableat4oCforweeks.
DilutionsofsICAM-1standards,512ng/ml->2ng/ml,serial2folddilutionsin1%BSA/PBS.- 512ng/ml
- 256ng/ml
- 128ng/ml
- 64ng/ml
- 32ng/ml
- 16ng/ml
- 8ng/ml
- 4ng/ml
- 2ng/ml
Procedure
- DiluatemAbR6.5(orCL203)to10ug/mlinPBS.Add50uldilutedantibodytowells.Incubate37oCfor1h,orO/Nat4oC.
- Wash4XwithPBS.
- Blockremainingprotein-bindingsitesbyadding200ul/wellof1%BSAinPBS.Incubate30minat37oC.
- Wash4XwithPBS.
- Add50ulofculturesupernatantcontainingsecretedICAM-1.IncludepositivecontrolwellsofpurifiedICAM-1rangingfrom512ng/mlto2ng/ml.Incubate30minat37oC.
- Wash4XwithPBS.
- Add50ul/wellBiotinylatedR6.5dilutedin1%BSA/PBStoafinalconcentrationof2ug/ml(1:1000dilutionofstock).Incubate30minat37oC.
- Wash4XwithPBS.
- Add50ul/wellHP-streptavidindilutedaccoridngtomanufacturersinstructions.Currentdilutionis1:2000in1%BSA/PBS.Incubate30minat37oC.
- Wash4XwithPBS.
- Wash1XwithSubstratebufferwithoutsubstrate.
- Add200ul/wellSubstratebuffer+ABTSSubstrate.
- Incubateatroomtemperatureuntilsufficientcolordevelops.Maybeasrapidas2minoraslongas20min.
- Readabsorbanceat414nm,blankingagainstnegativecontrolwell.
ExpectedResults
WegetourbestcurveswhenthemaximumAbs.forthe512ng/mlstandardis<1.500,butlessthan1.800.
References
相关仪器试剂设备
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