| 摘要: |
| [目的]研制快速、灵敏、特异的恩诺沙星残留检测ciELISA试剂盒(ENR-Kit),为动物源性食品中恩诺沙星(ENR)残留的快速免疫学检测奠定基础.[方法]通过细胞融合技术制备恩诺沙星单克隆抗体(ENR-mAb)杂交瘤细胞株,体内诱生腹水法生产ENR单抗,应用ENR mAb研制ENR残留间接竞争ELISA(ciELISA)快速检测试剂盒,并对其灵敏度、半数抑制浓度(IC50)、特异性、准确度和基质效应性进行检测.[结果]筛选出2株杂交瘤细胞,其单抗亚类均为IgGl亚型.4G1-B3杂交瘤细胞株的ENR mAb间接ELISA效价为1∶1.024×106,亲和常数(Kα)为9×1010L/moL.ENR-Kit的线性检测范围为0.05～48.75μg/L,灵敏度0.05μg/L,半数抑制浓度(IC50)为1.31μg/L,与环丙沙星的交叉反应率(CR)为0.02%,与其他化合物的交叉反应率(CR)均<0.01%.ENR-Kit对牛奶样,鱼肉样和鸡肉样中ENR的平均添加同收率分别为96.49%,86.95%和84.13%,平均变异系数均<10%,不同基质对ENR-Kit检测结果影响小.[结论]研制的ENR-Kit具有快速、敏感、特异、简便等特点,适合ENR残留快速检测的推广应用. |
| 关键词: 恩诺沙星 杂交瘤细胞 单克隆抗体 快速检测试剂盒 |
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| 基金项目:国家科技支撑计划? |
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| Preparation of monoclonal antibody and development of ciELISA kit for rapid detection of enrofloxacin |
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| Abstract: |
| 【Objective】 The study was done to develop a ciELISA kit to detect enrofloxacin residue. 【Method】 Balb/c mice were immunized with BSA-ENR.Hybridoma lines secreted monoclonal antibody against ENR were generated with cell fusion.ENR mAb was generated by inducing ascites in mice.A ciELISA kiting for detect ENR (ENR-Kit) was developed with ENR mAb and its sensitivity, IC50,veracity,specificity and stability were tested respectively. 【Result】 Two hybridoma lines were filtered and the best one was 4G1-B3,which secreted ENR mAb with indirect ELISA titers of 1∶1.024 ×106 in ascites.Isotype of the two mAb was IgG1.ENR mAb had a high affinity constant (Ka) of 9×1010L/mol.The ENR-Kit had a linear detection ranging from 0.05 to 48.75 μg/L,the sensitivity of 0.05 μg/L and a good sensitivity with an IC50 of 1.31 μg/L to ENR,0.02% cross-reactivity to Ciprofloxacin and less than 0.01% cross reactivity to other compounds.The recoveries of ENR spiked in milk,fish and chicken were 96.49%,86.95% and 84.13% respectively.The coefficient variation was below 10%. 【Conclusion】 The ENR-Kit is characterized with rapidity,sensitivity,specificity and briefness,and can be used for the rapid detection of ENR residues in animal food. |
| Key words: enrofloxacin hybridoma line monoclonal antibody ciELISA rapid detection kit |
