抗人Cu／Zn SOD单克隆抗体的制备与鉴定.pdf

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1、西部医学2010年4月第22卷第4期MedJWestChina，April2010，Vo1．22，No．4·603·抗人Cu／ZnSOD单克隆抗体的制备与鉴定蒋小梅，廖婷婷，章崇杰，张晨阳，魏大鹏(四川大学基础医学与法医学院免疫学教研室，四川成都610041)【摘要】目的制备抗人Cu／ZnSOD单克隆抗体并对其生物学特性进行鉴定。方法以人Cu／ZnSOD作为免疫原免疫Balb／c小鼠，取其脾细胞与SP2／0细胞融合，经多次筛选及克隆化，建立可稳定分泌抗人Cu／ZnSOD单克隆抗体的杂交瘤细胞株。用ELISA、斑点ELISA、WesternBlot、免疫组化鉴定单克隆抗体的特性，并测定其效价

2、、亚型及相对亲和力。结果筛选到一株可稳定分泌抗人Cu／ZnSOD单克隆抗体的杂交瘤细胞株4H1OH4B12，亚型鉴定重链为IgG2a，轻链为kappa链。ELISA法测定腹水效价为1；256，000，抗体相对亲和力为1．101×10mol／L。斑点ELISA、WesternBlot显示抗体能特异识别免疫原、人肝细胞株HL-7702和人红细胞中的天然Cu／ZnSOD；不与鼠Cu／ZnSOD和人MnSOD发生交叉反应。免疫组化进一步显示了人Cu／ZnSOD均匀分布于细胞的胞浆中。结论成功制备了抗人Cu／ZnSOD单克隆抗体，为进一步研究其与腹主动脉瘤的关系奠定了基础。【关键词】人Cu／ZnSO

3、D；单克隆抗体；腹主动脉瘤【中图分类号】R-331【文献标识码】A【文章编号】1672-3511(2O10)O4一O6O3一O5PreparationandidentificationofmonoclonalantibodyagainsthumanCu／ZnSODJIANGXiao-mei，LIAOTing-ting，ZHANGChen‘yang，etal(DepartmentofImmunology，SchoolofPreclinicalandForensicMedicine，SichuanUniversity，Chengdu610041，China)[Abstract]Objectiv

4、eToprepareandidentifythemonoclonalantibody(MAb)againsthumanCu／ZnSOD．Meth·odsBALB／CmicewereimmunizedwithimmunogenhumanCu／ZnSOD．Thespleencellsoftheimmunizedmicewerei—solatedandfusedwithSP2／0cells．Aftersevera1roundsofdetectingandcloning，ahybridomacel1linesecretinganti-hu—manCu／ZnSODMAbwasobtained．Its

5、specificitywasevaluatedwithindirect-ELISA，spot—ELISA，Westernblotandimmunohistochemistry．Thetiter，immunoglobulinsubtypeandaffinityoftheMAbweremeasured．Results0necelllineofhybridomanamed4HIOH4BI2wasobtained，whichwasidentifiedthattheheavyandlightchainsofanti-humanCu／ZnSODMAbwereIgG2aandK，respectively

6、，withhighaffinityof1．101×10。L／molandhightiterof1：256，000．Thespot-ELISA，WesternblotrevealedthattheMAbwasspecificallyagainsttheimmunogenandnativeproteinsex—pressedinHL-7702cells，humanhematidsanddidn’treactwithratCu／ZnSODorhumanMnSOD．Thecel1immuno-histochemistryprovedthattheMAbcouldrecognizethehumanC

7、u／ZnSOD1ocatedinkytoplasmofHL-77O2cells．Con—clusionThesuccessinmouseanti—humanCu／ZnS0DMAbpreparationprovidesthebasisforfurtherstudyofAAA．[Keywords]Human；Cu／ZnSOD：Monoclona1antibody)AAA超氧化物歧化酶(SOD)是一类广泛存在于生物行使功能起到