恩诺沙星单克隆抗体的制备及ciELISA试剂盒的研制  

Preparation of monoclonal antibody and development of ciELISA kit for rapid detection of enrofloxacin

作　　者：赵银丽[1,2] 王建华[1] 王自良[3,4] 滕蔓[3] 刘庆堂[3] 邓瑞广[3] 范国英[4] 张改平[3] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]河南工业大学生物工程学院,河南郑州450001 [3]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [4]河南科技学院动物科学学院,河南新乡453003

出　　处：《西北农林科技大学学报（自然科学版）》2009年第2期33-39,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家科技支撑计划项目(2006BAK02A21)

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（ENRmAb）杂交瘤细胞株，体内诱生腹水法生产ENR单抗，应用ENRmAb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IgG1亚型。4G1-B3杂交瘤细胞株的ENRmAb间接ELISA效价为1：1.024×10^5，亲和常数（Ka）为9×10^10L／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的平均添加回收率分别为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。[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, ICso ,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 × 10^6 in ascites. I-sotype of the two mAb was IgG1. ENR mAb had a high affinity constant （Ka） of 9 × 10^10 L/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 ICso 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 84.13μ 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.

关 键 词：恩诺沙星 杂交瘤细胞 单克隆抗体 CIELISA 快速检测试剂盒 

分 类 号：S859.84[农业科学—临床兽医学]