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为了研制氯霉素单克隆抗体,建立氯霉素的简便有效的检测方法。采用碳二亚胺(EDC)方法制备氯霉素免疫抗原和包被抗原,经SDS-PAGE凝胶电泳,三硝基苯磺酸(TNBS)法鉴定表明偶联成功。采用制备的抗原CAP-HS-BSA((CAP-HS,氯霉素琥珀酸酯;BSA,牛血清白蛋白)免疫Balb/c小鼠,通过杂交瘤技术获得了2株抗氯霉素的杂交瘤细胞株,细胞株体外传代和冻存复苏后抗体分泌稳定。经体内诱生法产生腹水,ELISA检测纯化腹水的效价为1∶106。抗体的亲和常数分别为:1.13×1010L/mol,1.41×1010L/mol。ELISA检测显示这2株单克隆抗体与氯霉素琥珀酸交叉反应率为300%,与其他抗生素及结构类似物的交叉反应小,表明该单抗可满足建立免疫学检测方法的需要和开发应用的要求。
Abstract：In order to prepare momclonal antibody(Mab) against chlormphenicol(CAP) for CAP detection,CAP was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to prepare immune antigen and detection antigen by the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) method respectively,then the artificial antigens were detected by SDS-PAGE,and the residual amount of ε-amino groups was determined by the TNBS method.The result showed it was successfully synthesized.Balb/c mice were immunized with conjugated CAP-HS-BSA,and two strains of hybridomas which secret high titer antibodies against CAP were cloned and then ascitic fluids were made,the titers of ascites were 1×106 by ELISA and the affinity constant was 1.13×1010 L/mol and 1.41×1010 L/mol,respectively.The number of chromosome of the hybridoma cell lines was 90～104.The cross reaction rate of Mab against CAP and CAP-HS was 300%,and the Mab did not have cross reactivity with other antibiotics.The result showed the Mab can be used for the detection of CAP.
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基本信息:
DOI：10.16441/j.cnki.hdxb.2010.03.016
中图分类号:R392.11
引用信息:
[1]方伟,姜有声,黄宣运,等.氯霉素单克隆抗体的研制及其特性分析[J].中国海洋大学学报(自然科学版),2010,40(03):90-94.DOI:10.16441/j.cnki.hdxb.2010.03.016.
基金信息:
十一五国家科技支撑计划(2006BAD03B04);; 上海市科技兴农重点攻关项目(6660106477);; 上海市重点学科建设项目(Y1101);; 现代农业产业技术体系建设专项资金;; 现代农业产业技术体系专项(nycytx-49-17);; 公益性行业(农业)科硕专项经费——对虾现代产业技术体系研究与建立(nyhyzx07-042)资助
2010-03-15
2010-03-15