抗恩诺沙星单克隆抗体杂交瘤细胞株的筛选及竞争ELISA试剂盒的研制

SCREENING OF HYBRIDOMA SECRETING MCAB TO ENROFLOXACIN AND DEVELOPMENT OF COMPETITIVE ELISA ENR-KIT

利用合成的完全抗原(BSA-ENR)免疫小鼠,通过细胞融合技术和间接竞争ELISA筛选出了能分泌恩诺沙星单克隆抗体的杂交瘤细胞株,以此为基础研制恩诺沙星残留检测的竞争ELISA试剂盒(competitive ELISA ENR-Kit),并测定其性能。结果表明:筛选出的2株杂交瘤细胞,单抗亚类均为IgG1型,其中4G1-B3株的ENR mAb间接ELISA效价为1:1.024×106,亲和常数(Ka)为9×1010L/mol;竞争ELISA试剂盒的线性检测范围为0.05~101.6μg/L、灵敏度0.05μg/L、半数抑制浓度(IC50)1.1μg/L,与其他化合物的交叉反应率(CR%)均小于0.01%,鸡肉样、鸡肝样、鱼肉样和牛奶样的平均添加回收率分别为81.5%8、7.6%、84.3%和95%,不同基质添加恩诺沙星标准品做竞争ELISA对ENR-Kit检测结果影响小,试剂盒保存期6个月以上。结果说明该竞争ELISA试剂盒具有快速、敏感、特异、简便等特点,适用于ENR残留的快速检测。

Immunogen antigen was prepared by active ester method coupling enrofloxacin to BSA. Balb/c mice were immunized with BSA-ENR. Hybridoma lines that secreted antibody against ENR were generated with cell fusion and Screened of hybridoma was screened by indirect competitive ELISA. ENR mAb was generated by inducing ascites in mice. A competitive ELISA kit for detect ENR was developed with ENR mAb and its traits were tested. 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 IgG~ . ENR mAb had a high affinity constant （Ka） with 9 x 10X^L/mo｜. The ENR-Kit had the linear detection range of 0.05 N 101.6ttg/L, the sensitivity of 0.05 /~g/L and a good sensitivity with an ICs0 of 1.1/~g/L to ENR, cross-reactivity to other compounds less than 0.01%. The average recoveres of ENR spiked in chicken, liver, fish and milk were 81.5%, 87.6 %, 84.3 % and 95 % respectively. The coefficient variation was below I0%. The competitive ELISA ENR-Kit possesses characters of rapidity, sensitivity, specificity and briefness, which is to be used for the rapid detection of ENR residues in animal food.