Numeroustechniqueshavebeendevelopedtoprepareimmunoliposomesbasedonthenucleophilicreactivityoffreeaminegroupsofproteinsorpeptides.OneofthemostpopularandcommonlyusedmethodsistocovalentlycouplefreecarboxylicgroupstoprimaryaminesthroughactivationofthecarboxylgroupswithEDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide).EDC,whichisaso-calledzero-lengthcrosslinkingagent,reactswiththecarboxyltoformanaminereactiveintermediate(O-acylisourea).TheproducedO-acylisoureacanbeeasilydisplacedbynucleophilicattackfromtheprimaryaminogroupsinthereactionmixture.However,thisintermediateisunstableandhydrolyzedinaqueoussolutions.Inordertopreventtheintermediatehydrolysis,sulfo-NHS(N-hydroxysulfosuccinimide)isaddedtoEDCtoproduceasignificantlymorestableandmoresolubleactiveintermediate(NHSester).

Consequently,theimmunoliposomesarepreparedbyatwo-stepcouplingprocedure:first,activatingthefreecarboxylgroupofthelinkerlipidincorporatedintheliposomeswithEDCandsulfo-NHS,andthencovalentlyconjugatingtheantibodiestothelipidsthroughdisplacementofsulfo-NHSgroupsbyantibodyamines,asdepictedbelow.EDC/sulfo-NHScouplingreactionsarehighlyselectiveandhighlyefficient,andtheBIOLOGicalactivityoftheproteinorpeptideispreserved.

ImmunoFluor™-CarboxylicAcidisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalatedproducts)andalsoImmunoFluor™productssuitableforothertypesconjugationmethodsseehere.