
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.
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采用抗-HBe抗体包被反应板,加入校准品及被测样本,同时加入定量HBeAg中和抗原,经过振荡孵育,洗板后再加入铕标记的抗-HBe,若标本中抗-HBe浓度高,HBeAg将被大量中和,使最后形成的抗-HBe-HBeAg-铕标记抗-HBe复合物减少。增强液(β-NTA)将标记在抗体上的Eu3+解离到溶液中,Eu3+和增强液中的有效成分形成高荧光强度的螯合物,荧光强度和样本中的抗-HBe浓度成反比。