微孔板蛋白质芯片技术应用于单克隆抗体分型研究3

ChinaBiotechnology,2007,27(6):566031111211133(1/201203)(2201203),122,ELISA,,ELISA;ELISA,,,,Q816:2007202201:20072032053(B)(05XDB1414),863(2006AA020704)33,:[e-mail][15],[68],ELISA,ELISA,,,[9],,122,ELISA,,,11.11.1.1122,,pH7.0,50mmol/LPBS,,pH7.0,50mmol/LPBS120001.1.2(ISO22)(HRP)(A9917)(TMB,T0565)Sigma,(MultiScreen962WellPlates,MAIPN45)Millipore,CartesienAD5000,Perfection640U(Epson)1.21.2.12007,27(6):ISO226,pH7.0,50mmol/LPBS33,,1(a),633,,1(b)1.2.2Millipore70%,PBS,,25nl,1(c)371h,41a:;b:;c:96Fig.1Thesketchmapandthepictureofthemicro2wellproteinarray1.31.3.1,50l1h,(PBST,0.1%20)3,3min;50lpH7.0,50mmol/LPBS13000HRP,0.5h,PBST3,3min;30lTMB,1min,1.3.2ELISAELISASigmaISO22,ELISA(captureELISA),100l22.1,4(mAb201,mAb202,mAb204,mAb207)110,140,1160,1640,12560,110240,IgM,2(a),1,1:10,6,140,IgG2bIgG2b,120,1Table1Theresultsoftheselectionoftheisotypingantibodyconcentrationusedinthemicro2wellproteinarrayIgG1IgG2aIgG2bIgAIgMIgG31101401401101401401401401401401101401401401160140140211014014014016402222140222214021402222212560222222222222222214022222110240222222222222222222222222mAb202mAb201mAb204mAb204mAb207mAb20775ChinaBiotechnologyVol.27No.620071160,5110140,12061160,,11002.2122,32(b),2,7IgG1,3IgG2a,1IgG2bIgA,1IgMIgG3;252.3ELISAELISA,ELISA(2)2(a):IgM;(b):1:mAb201;2:mAb207;3:pAb202Fig.2Theresultsofthehybridizationofthemicro2wellproteinarray2Table2TheisotyperesultsofthemonoclonalandpolyclonalantibodiesELISAmAb201LMO2IgG2aIgG2amAb202MYCIgG1IgG1mAb203GNAZIgG2aIgG2amAb204PLAUIgG2bIgAIgG2bIgAmAb205Paf2AHIgG1IgG1mAb206Paf2AHIgG2aIgG2amAb207Paf2AHIgMIgG3IgMIgG3mAb208MLPIgG1IgG1mAb209MLPIgG1IgG1mAb210MLPIgG1IgG1mAb211MLPIgG1IgG1mAb212MLPIgG1IgG1pAb201PECIIgG1,IgG2a,IgG2b,IgM,IgG3IgG1,IgG2a,IgG2b,IgM,IgG3pAb202MLPIgG1,IgG2a,IgG2b,IgM,IgAIgG1,IgG2a,IgG2b,IgM,IgA3,6Sigma,6(IgG1,IgG2a,IgG2b,IgG3,IgA,IgM),HRPIgG(Fab),,4(mAb201,mAb202,mAb204,mAb207),mAb201IgG2a,mAb202IgG1,mAb204IgG2bIgA,852007,27(6):mAb207IgMIgG3,46,1:20122ELISA,223,100%,2,96PVDF,ELISA,19648(),1ELISA968()ELISA(3),,,,,,1:100,,,,3ELISATable3ThecontrastbetweentheELISAandthearraymethodappliedinoneantibodyisotypedELISA200nl2.5nl1200ml1l0.4l0.03l3h1.5h96()848,,1h29633,96,42,,,[1]DeWildtRM,MundyCR,GorickBD,etal.Antibodyarraysforhigh2throughputscreeningofantibody2antigeninteractions,NatBiotechnol,2000,18(9):989994[2]GloklerJ,AngenendtP.Proteinandantibodymicroarraytechnology,JChromatogrBAnalytTechnolBiomedLifeSci,2003,25(797):229240[3]MichaudGA,SalciusM,ZhouF,etal.Analyzingantibodyspecificitywithwholeproteomemicroarrays,NatBiotechnol,2003,21(12):15091512[4]Bensmail1H,HaoudiA,Postgenomics:Proteomicsandbioinformaticsincancerresearch.JournalofBiomedicineandBiotechnology,2003(4):217230[5]SternM,HerrmannR.Overviewofmonoclonalantibodiesincancertherapy:presentandpromise,CriticalReviewsinOncologyHematology,2005,54(1):1129[6]ChambersRS.High2throughputantibodyproduction,CurrOpinChemBiol,2005,9(1):4650[7]DeMasiF,ChiarellaP,WilhelmH,etal.Highthroughputproductionofmousemonoclonalantibodiesusingantigenmicroarrays.Proteomics,2005,5(16):40704081[8]TraggiaiE,BeckerS,SubbaraoK,etal.AnefficientmethodtomakehumanmonoclonalantibodiesfrommemoryBcells:potentneutralizationofSARScoronavirus.NatMed,2004,10(8):871875[9]MacBeathG,SchreiberSL.Printingproteinsasmicroarraysforhigh2throughputfunctiondetermination.Science,2000,289(5485):1760176395ChinaBiotechnologyVol.27No.62007Micro2wellProteinArrayTechnologyAppliedinMonoclonalAntibodyIsotypingSONGKai1ZHOUJia2jing1YESai1PENGHai2lin1WANGSheng2nian2XIAOHua2sheng1ZHAOGuo2ping1ZHANGQing2hua1(1NationalEngineeringCenterforBiochipatShanghai,ShanghaiBiochipCo.LTD.,Shanghai201203,China)(2ShanghaiHuaguanBiochipCo.LTD.,Shanghai201203,China)AbstractMonoclonalantibodyisotypingmicro2wellproteinarraywasdesignedandestablished.TwelvemonoclonalantibodiesandtwopolyclonalantibodieswereisotypedbytheisotypingproteinarrayandELISArespectively.TheresultsshowedthattherewasnodifferencebetweenproteinarraymethodandELISAintheapplicationofisotypingmonoclonalandpolyclonalantibodies.AndfewersampleandmaterialswereusedandtheefficiencywasimprovedinproteinarraymethodcomparedwithELISA.Asaresult,theantibodyisotypingproteinarraycouldbeusedasasensitive,rapid,directionalandeconomicaltoolapplyinginhighthroughputmonoclonalantibodygeneration.KeywordsProteinarrayMonoclonalantibodyIsotypeBrock(,)()[]M.T.978-7-03-018857-1/Q.1841:168.0020075Brock(),,,:,,,,,()[]978-7-03-017977-7/Q.1778:68.0020074MethodsinMolecularBiology204(FISH)(SKYM-FISHCGHFISHCGH),,,978-7-03-018527-3/Q.1822:98.002007350,-460():10071716,::010-64034622()