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TheindirectELISAisusedprimarilytodeterminethestrengthand/oramountofantibodyresponseinasample,whetheritisfromtheserumofanimmunizedanimalorthecellsupernatantfromgrowinghybridomaclones.
Procedure
- Allincubationsaredoneasfollows:Coverwithplatesealertapeorplaceinasealedboxcontainingawetpapertowelandincubatefor2hoursatroomtemperature.Forthefourcontrols:2arenegativecontrolsusingpre-bleedsofthesameconcentrationasyourstaringdilutionof1°antibody;thethirdisanegativecontrolwhereno1°antibodyisadded,justblockingbufferatthisstep;andthefourthisapositivecontrol,eitherfromapreviouslypositivebleedorcellsupernatant,oryoucanlaydown1°antibodyasantigen.
- Ina96-wellELISAplate(NuncMaxiSorpisbest),add100ngofantigenin50µLineachwellyouwillbeusingforyourtest,aswellasfourcontrolwells.Theperimeterwellsontheplatearegenerallynotused,astheytendtogivepoorresults.Incubate.
- Dumpouttheantigensolutionandadd100µLofblockingbuffer(1BSA,0.1MKPi,0.1Tween-20,0.02thimerisol,pH7).IfyourcarrierproteinforinjectionwasBSA,thensubstitute1non-fatdrymilkfortheBSA.Incubate.Theblockingstepincubationcanbealsodoneat4°Covernight.
- Dumpouttheblockingbufferandbangtheplateupsidedownonsomepapertowelstoremovealltheliquid.Wash3timeswithwashbuffer(0.1MKPi,0.05Tween-20,pH7),shakingthewashoutvigorouslyeachtime.Againbangouttheresidualwashbuffer.
- Add50µL/wellofyour1°antibody.Forscreeninghybridomas,thiswillbecellsupernatant.Forobtainingatiteronserumfromanimmunizedanimal,youwillneedtoperformserialdilutionsoftheseruminblockingbufferintheplate.1:1serialdilutionsaredonebyplacing100µLinthefirstcolumnofyourplateofyourstartingdilutionofserum(1:499inblockingbufferisusuallyagoodstartingpoint).Thenplace50µL/wellofblockingbufferdownallthewellsremainingintherowsyouareusing.Nowpipetout50µLfromthefirstwell(withyourstartingdilution)andplaceinthenextwellintherow.Mixbypipetingthesolutionupandown,andthentransfer50µLofthissolutiontothenextwellandagainmix.Continuethesedilutionsdowntherowuntilthelastwell,whereyouremove50µLandthrowaway.Incubate.
- Wash3timesasbefore.
- Add50µL/wellofa1:1999dilutioninblockingbufferofHRP-labelled2°antibodythatisdirectedagainstthespeciesofyourprimaryantibody(anti-mouseformonoclonalsandmouseserum,anti-rabbitforpolyclonalantibodiesraisedinrabbits).Incubate.
- Wash3timesasbefore.
- Add100µL/wellofABTShorserADIshperoxidasesubstrate.Incubateatroomtemperaturefor5-20minutes,dependingontherateofcolordevelopment.Keepthetimeidenticalforsubsequentcomparisonsoftiter,andforhybridomascreeninggothefull20minutes.
- Add100µL/wellstopsolution(0.5MOxalicAcid).
- Readabsorbanceat414nminanELISAreader.
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