磺胺母核单克隆抗体的制备及其免疫学特性研究  

Immunological Characteristics Research and Preparation of Monoclonal Antibodies of Sulfanilamide Mother Nucleus

作　　者：杜玉玲[1,2] 常迪[2] 吴国英 陆彦[2] 王晶[2] 吴国娟[2] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]北京农学院动物科学技术学院,北京102206 [3]吉林省长春市双阳区妇幼保健所吉林长春130600

出　　处：《中国食品学报》2013年第12期139-145,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31172362);北京市属高校人才强教深化计划项目(PHR201106219)

摘　　要：制备磺胺母核(SH)单克隆抗体(Monoclonal antibody,mAb),并对其免疫学特性进行检测。方法:采用混合酸酐法将本实验室合成的磺胺类药物母核结构物(SH)偶联于牛血清白蛋白(BSA)和卵清蛋白(OVA)上,合成免疫原BSA-SH和包被原OVA-SH,并用紫外扫描(UV)、SDS-PAGE法进行鉴定。用BSA-SH免疫Balb/c小鼠,间接ELISA和间接竞争ELISA法筛选细胞融合备用鼠;应用杂交瘤技术建立SH mAb细胞株,用体内诱生腹水法制备SH mAb,对SH mAb的效价、敏感性和特异性等免疫学特性进行鉴定。结果:BSA-SH人工抗原制备成功,SH与BSA的分子结合比约为11.6∶1。筛选出5G1、5G2和8D2 3株敏感特异性的杂交瘤细瘤细胞,其细胞培养上清液效价分别为1∶6.40×102,1∶1.28×103和1∶3.20×102,腹水效价分别为1∶2.56×105,1∶5.12×105和1.28×105。5G2株对SH的半数抑制浓度(IC50)为82 ng/mL。纯化后抗体对磺胺间甲氧嘧啶(SMM)、磺胺对甲氧嘧啶(SMD)、磺胺甲基嘧啶(SM)、磺胺间二甲氧嘧啶(SDM)、磺胺醋酰(SCT)、磺胺氯吡嗪钠(SCZ)、磺胺噻唑(ST)、磺胺甲噁唑(SMZ)、磺胺苯吡唑(SPH)、磺胺二甲嘧啶(SM2)、氨苯磺胺(SSF)和磺胺喹噁啉(SQ)的交叉反应率分别为108%,86%,105%,85.6%,72%,26%,32%,54.6%,43%,4.6%,0.5%和76%。结论:通过试验获得了抗磺胺母核SH高效价、敏感、特异的mAb,为研制磺胺药多残留检测试剂盒和胶体金免疫层析试纸条奠定了基础。Objective：The monoclonal antibodies against Sulfanilamides were prepared and their immunological charac-teristics were identified.Method：Mixed-anhydride was used to conjugate SH that a hapten N-sulfanily-4-aminobenzoic was synthesized by our laboratory,it including the structure of sulfanilamide common to all sulfanmides and with the spacer arm of benzene ring,to carrier protein BSA and artificial antigen was obtained.The coating antigen OVA-SH was obtained in the same way.Ultra-violet（UV） and SDS-PAGE were used to identify SH artificial antigen.Balb/C mice were immunized with BSA-SHandthe titre of polyclonal antibody（pAb） was detected by indirect ELISA and indirect com-petitive ELISA.The hybridoma lines that secrete SH mAb were established with monoclonal antibody hybridoma technolo-gy.The immunological characteristics such as titer,sensitivity and specificity of the mAb were studied.Results：SH artifi-cial antigen was synthesized successfully and its conjugation ratio of SH to BSA was about 11.6∶1.Three hybridoma cell lines of 5G1、5G2 and 8D2 were screened for specificity to SH.The indirect ELISA titer of the mAb were 1∶6.40×10^2,1∶ 1.28×103,1∶3.20×10^2in supernatant and 1∶2.56×10^5,1∶5.12×10^5,1.28×10^5in ascites.The mAb of 5G2 showed good sensi-tivity with an IC50 of 82 ng/mL to SH.The crossreactive rates of the mAb after purified toSulfamonomethoxine（SMM）,Sulfametoxydiazine（SMD）,Sulfamerazine（SM）,Sulfadimethoxine（SDM）,Sulfacetamide（SCT）,Sulfaclozine sodium monohy-drate（SCZ）,Sulfathiazole（ST）,Sulfamethoxazole（SMZ）,Sulfaphenazole（SPH）,Sulfamethazine（SM2）,Sulfasulfonamide（SN） and Sulfaquinoxaline（SQ） were 108%,86%,10^5%,85.6%,72%,26%,32%,54.6%,43%,4.6%,0.5% and 76%.Conclusion：SH mAb of high-titer,sensitivity and specificity has been generated,therefore,the experiment laid the foundation for the study of ELISA kit and Colloidal gold immunochromatography test strip fordetecting the residues of Sulfonamides.

关 键 词：磺胺类药物 母核 单克隆抗体 免疫学特性 多残留检测 

分 类 号：S859.84[农业科学—临床兽医学]