抗HCCR单克隆抗体的制备及其鉴定.pdf

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1、实用临床医药杂志2·6·JournalofClinicalMedicineinPractice⋯0l2l川l6卷LI3jfJjJ抗HCCR单克隆抗体的制备及其鉴定郝波，林艳，张国新，施瑞华(南京医科大学第一附属医院消化实验室，江苏南京，210029)摘要：目的制备抗人宫颈癌癌基因(HCCR)单克隆抗体(mAb)，并进行鉴定。方法以HCCR重纰蛋n免疫IB／c小鼠，取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2／0进行细胞融合，常规筛选杂交瘤细胞。腹水j秀导法制箭抗人tlCCR单克隆抗体，并经蛋白A亲合层析进行纯化。用Westernblot和免疫荧光等

2、方法枪测ttCCRmAb的特异H-刚纯化HCCR蛋白进行包被，建立间接ELISA方法，检测系列稀释的HCCRmAb，确定其灵敏度。结果筛选flj2株分泌抗人H(：(：I{m的杂交瘤细胞株。Westernblot结果表明抗人HCCR抗体能与HCCR蛋白特异性结合，免疫荧光结果示癞HepG2细胞rf1胞浆和胞膜均呈阳性染色。问接ELISA方法检测HCCRmAb的灵敏度可达到1g／L。结论成功删嵛外鉴定了特片性抗人HCCRmAb的杂交瘤细胞株，为进一步深入研究HCCR的生物学特征和临床应用打下基础。关键词：HCCR；单克隆抗体；杂交瘤中图分类号：R446．

3、62文献标志码：A文章编号：1672—2353(2012)03—0006—04PreparationandidentificationofmonoclonalantibodyagainsthumancervicalcanceroncogeneHAOBo，LINYan，ZHANGGuo—xin，SHIRui—hua(TheFirstHospitalAffiliatedtoNanjingMedicalUniversity，Nanjing，Jiangsu，210029)ABSTRACT：ObjectiveToprepareandidentifymonocl

4、onalantibodyagainsthumancervicalcanceroncogence(HCCR)．MethodsPurifiedrecombinantHCCRproteinwasusedasantigentoimmunizeBALB／cmice．TheisolatedspleencellsfromtheimmunizedmicewerefusedwithSP2／0．MonoclonalantibodiesagainstHCCRwerepreparedbynormalhybridomatechnology．PurifiedmAbwasprep

5、aredbyproteinAchromatography．ToidentifythecharacteristicsofHCCRmAbs，Westernblotandimmunofluorescencemethodwereemployed．TheindirectELISAWasestablishedwiththeporifledrecombinantHCCRprotein．andusedtodetectthesensitivityofmAbsagainstHCCR．ResultsTwohybridomacelllineswhichcouldsecret

6、eanti—humanmAbagainstHCCRwereestablishedbyhybridomatechnique．SpecificityofmAbagainstHCCRwasassessedbyWesternblot．Immunofluores—cenceshowedthatHCCRpositivestainingoccurredinthecellhyalomitomeandmembraneofhepaticcarcinomaHepG2．TheindirectELISAwasestablishedwiththepurifiedHCCRprot

7、ein，andwiththisindirectELISA，aslowas1L0ffunctionalanti—humanmAbagainstHCCRcouldbedetected．ConclusionHumanHCCRhybridomacelllinesandtheirsecretedmAbshavebeensuccessfu11v0b—rainedandidentified．whichlaysasolidfoundationtofurtherstudythebiologicMcharacteristicsandclinicalapplication

8、sofHCCR．KEYWolIS：humancervicalcanceroncogence；monoclon