双抗夹心elisa定量检测血液中rhu ifn-ω的分析-quantitative determination of rhu ifn - ω in blood by double antibody sandwich elisa.docxVIP

ABSTRACTAsarecentlyfoundinterferonwithbroad-spectrumantivirusfunction,enhancementfacilitationonimmunologicalfunctionanddepressanteffectoncellgrowth,recombinantinterferon-ω(IFN-ω)hasimportantpotentialindiseasetreatment.IthasbeencompletelyinvestigatedinaspectsofrecombinantHumaninterferon-ω(rHuIFN-ω)geneexpression,purificationandnature.Theinvestigationinclinicalpharmacokineticsandpharmacodynamicsisinprogresscurrently.Toachieveit,itbecomesnecessarytoestablishadependableassaymethod.Comparedtovarieskindsofmethods,enzymelinkedimmunosorbentassay(ELISA)isfoundtobeaccurate,sensitive,specificandsimple.double-antigensandwichELISAtoassayrHuIFN-ωinbloodonquantitywasestablishedinthisstudy.Theresearchworkisasfollowing:①preparationandpurificationofrabbitpolyclonalantibodyofrHuIFN-ωNew-ZealandalbinorabbitswereimmunizedwithrHuIFN-ωandFreundadjuvanttoprepareantiserum.Theantiserumwaspurifiedbytri-degreeprecipitationwithsaturatedammoniumsulfate(50%,40%,33%),andthendiethylaminoethyl(DEAE)columnforfurtherpurification.Highpurepartwascollectedstepbystep.ItsantibodytiterwasdetectedbyindirectELISA,whileconcentrationbyLowyapproach.Finally,theantibodyforadouble-antigensandwichELISAwasobtained.②preparationofenzymelabeledantibodyThepurifiedantibodieswerecross-linkedwithHRP(horseradishperoxidase)byglutaraldehydedouble-stepapproachtogainenzymelabeledantibodies.ThentheenzymelabeledantibodieswerecollectedbySuperdex-200GECstepbystepandseparatedbyELISAactivityexperimentaslabeledandunlabeledantibodies,unlabeledenzymes.Sotheenzymelabeledantibodiesfordouble-antigensandwichELISAwereobtained.③theoptimalconditionfordouble-antigensandwichELISAandstandardcurveTheconditionoptimizedbycheckboardapproachis:antibodycoatingconcentration50μg/mL,enzymelabeledantibodydilution1:100.ThreestandardcurveswasestablishedforrHuIFN-ωinoriginalserum,5timesdilutedserumand10timesdilutedserum.ItwasindicatedthatwhentheconcentrationofrHuIFN-ωisintherangeof15pg/mL~480pg/mL,standardrHuIFN-ωconcentrationnaturallogarithmisfavorablylinearc