Polyclonal Antibodies
ImmunizationofMice:
Injectionsshouldbemadeatintervalsofatleasttwoweeks..Wehavebeenusingeitherofthetwofollowingadjuvantswithsuccess:FreundadjuvantandMPL+TDMadjuvant(RIBIImmunoChemResearch,Inc.),thelatterbeinglesshazardousthanthefirst.
1stinjection:15-50ugAntigenin200uladjuvant,injectedsubcutaneously.2ndinjection:2weekslater;boostwith15-50ugofAntigeninadjuvant.(Iftimepermits,boostagainamonthlater).7-10dayslaterbleedthemouseandtestserumusingtheassaywhichwillbeusedforscreening.Iftitreisnothighenough,boostagaintwoweeksafterpreviousboost.SerumPreparation:
Aftercollecting,bloodshouldbeallowedtoclotfor60min.at37°CorO.Nat4°C.Separatetheclotfromthesidesofthetube(ringing)usingapasteurPipette.Placeclotat4°CO.N.Spinat10000xgfor10min.at4°Ctoseparatetheserum.Serumcanbestoredat-20°CafteraddingGlycerolto50.Ifmonoclonalantibodiesaredesired,onceyouhavea\"good\"polyclonalandareliablescreeningmethodproceedtothenextstep.
Rabbits:
Rabbitsareimmunizedessentiallyasdescribedabove.Injections:
FirstinFreundscomplete100-200ug/1mlinjection,twosites(overeachshoulder).Second1monthlaterinFreundsINCOMPLETEasabove.Bleed2weekslater.Boost2weekslateragain(2+2weekcycle).
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