环丙沙星单克隆抗体的制备

Preparation of the monoclonal antibody against Ciprofloxacin

【目的】制备环丙沙星(Crprofloxacin,CPFX)单克隆抗体(monoclonal antibody,mAb),并对其免疫学特性进行鉴定,为畜产品CPFX残留的快速检测提供技术支持。【方法】采用碳二亚胺法(EDC),将载体蛋白牛血清白蛋白(Bovine serum albumin,BSA)和鸡卵白蛋白(ovalbumin,OVA)分别与CPFX偶联合成免疫抗原CPFX-BSA和包被抗原CPFX-OVA,经紫外(UV)扫描和聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定后免疫BALB/c小鼠,应用细胞融合技术筛选分泌CPFX mAb的杂交瘤细胞株,采用体内诱生腹水法制备CPFX mAb,并对其染色体核型、效价、敏感性和特异性等免疫学特性进行鉴定。【结果】UV和SDS-PAGE鉴定结果表明,人工抗原偶联成功;免疫的5只小鼠血清抗体效价均达到了1∶105左右,其中4号小鼠血清CPFX抑制效价较高且IC50最低(17.21μg/L),融合后筛选出1株敏感特异的杂交瘤细胞,命名为3D2。3D2细胞培养上清效价为1∶(1.28×103),腹水效价为1∶(1.6×105)。CPFX mAb对CPFX的IC50为9.95μg/L,对恩诺沙星的交叉反应率为128.39%,与其他同系的喹诺酮类抗生素的交叉反应率很低。【结论】获得了高效价、敏感、特异的抗CPFX mAb,为CPFX残留的快速检测奠定了技术基础。

【Objective】 The study was conducted to generate monoclonal antibodies against Ciprofloxacin（CPFX） and to identify its immunological traits for the detection of CPFX residue in livestock products.【Method】 The CPFX was conjugated to carrier protein BSA and OVA by EDC to obtain immunogen and coating antigen.The ultraviolet scanning and SDS-PAGE were used to identify CPFX artificial antigen.Then BALB/c mice were immunized with CPFX-BSA.The hybridoma lines that secrete monoclonal antibody against CPFX（CPFX mAb） were established through cell fusion.CPFX mAb was generated by inducing ascites in mice.The immunological characteristics such as Karyotype,titer,sensitivity and specificity of the mAb were characterized.【Result】 The ultraviolet scanning and SDS-PAGE showed that CPFX artificial antigen was synthesized successfully.Five BALB/c mice indirect ELISA titer against CPFX were all above 1∶105 and the IC50 of No.4 mice was the lowest（17.21 μg/L）.One hybridoma cell line of 3D2 was screened for specificity to CPFX after cell fusing.The indirect ELISA titer of the mAb was 1∶（1.28×103） in supernatant,1∶（1.6×105） in ascites.The mAb of 3D2 showed high sensitivity with an IC50 of 9.95 μg/L to CPFX.The rate of cross reaction to Enrofloxacin could reach 128.39%.There were low reactions to other fluoroquinolones antibiotics.【Conclusion】 The high-titer,sensitive and specific CPFX mAb has been generated,which laid a solid foundation for the detection of CPFX residue.