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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (18): 12-16.

所属专题： 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

环丙沙星单克隆抗体的制备及其免疫学特性鉴定

职爱民 刘庆堂 李青梅 杨苏珍 柴书军 赵东 张志杰 邓瑞广 张改平   

  • 收稿日期:2010-01-29 修回日期:2010-04-28 出版日期:2011-09-20 发布日期:2011-09-20

Generation and Immunological Traits of Monoclonal Antibody for Ciprofloxacin

  • Received:2010-01-29 Revised:2010-04-28 Online:2011-09-20 Published:2011-09-20

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摘要： 用EDC法将BSA和OVA分别和环丙沙星（Ciprofloxacin, CIP）偶联作为免疫原和包被原，并经紫外扫描 (UV Scan)、聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定。用合成的BSA-CIP免疫BALB/c小鼠，经过3次免疫后，用间接ELISA和阻断ELISA选择细胞融合备用鼠，选择高效价、敏感的小鼠进行抗原超强免疫；取其脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌CIP单克隆抗体的杂交瘤细胞株；用体内诱生腹水法制备CIP mAb，对CIP mAb的效价、敏感性和特异性等免疫学特性进行鉴定。聚丙烯酰胺凝胶电泳鉴定表明BSA-CIP人工抗原偶联成功；免疫的3只小鼠血清抗体效价均达到10-4；其中2号小鼠血清CIP抑制效价较高且IC50最低，达48.59μg/L，融合后筛选出Z3G12B3和Z2H8C10两株敏感特异的杂交瘤细胞，其两株细胞培养上清液效价为1:512，腹水效价为1:2.56×105，Z3G12B3株对CIP的IC50为2.47μg/L，与二氟沙星、沙拉沙星有小于0.03％的交叉反应性，与其他抑制物无交叉反应性。本试验获得了高效价、敏感、特异的抗CIP mAb，为CIP残留ELISA检测试剂盒和试纸条的建立奠定了坚实的基础。

关键词: 转rd29A基因国槐, 转rd29A基因国槐, NaCl胁迫, 生理指标

Abstract: The Ciprofloxacin (Ciprofloxacin, CIP) was conjugated to carrier protein BSA and OVA by EDC to obtain the artificial and coating antigen . The ultraviolet scanning and SDS-PAGE were used to identify Ciprofloxacin artificial antigen. BALB/c mice were immunized with BSA-CIP. The titer of polyclonal antibody (pAb) was detected by indirect ELISA and blocking ELISA after three times immunization. The high titer, sensitive and specific mouse was selected for cell fusing. The hybridoma lines that secrete CIP mAb were established with using monoclonal antibody hybridoma technology and the immunological characteristics such as titer, sensitivity and specificity of the mAb were characterized. SDS-PAGE results showed that CIP artificial antigen was synthesized successfully. Three BALB/c mice indirect ELISA titer against CIP were all above 10-4 and the IC50 of No. 2 mice was 48.59μg/L. Two hybridoma cell lines of Z3G12B3 and Z2H8C10 were screened for specificity to CIP after cell fusing, the indirect ELISA titer of the mAb were 1:512 in supernatant, 1:2.56×105 in ascites, the mAb of Z3G12B3 showed good sensitivity with an IC50 of 2.47μg/L to CIP, had less than 0.03％cross-reactivity with sarafloxacin and difloxacin, and no cross-reactivity to other compounds. The high-titer, sensitive and specific CIP mAb has been generated and laid a solid foundation for the CIP ELISA kit and strip.