玉米赤霉醇特异性单克隆抗体及其胶体金检测试纸的研制  

Development of Specific Monoclonal Antibodies and Colloidal Gold Test Strips for Zeranol

作　　者：刘媛 文孟棠 张霄 徐重新 杨晶祎 张留娟 王恒 何鑫 梁颖 郑勤 夏兴霞 刘贤金 

机构地区：[1]江苏省食品质量安全重点实验室/农业部食用农产品安全监控重点开放实验室,江苏南京210014

出　　处：《核农学报》2015年第6期1101-1107,共7页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(31201535);江苏省社会发展项目(BE2012750);江苏省农业自主创新基金CX(12)5042;948项目(2011-G513);江苏省自然科学基金(BK20130701)

摘　　要：为了研究国内制备的玉米赤霉醇单克隆抗体,与其同系物玉米赤霉烯酮交叉反应严重,实际检测存在假阳性的问题,本试验开展了玉米赤霉醇特异性识别单克隆抗体及其胶体金检测试纸的研制工作。试验采用玉米赤霉醇-16-羧丙基醚与牛血清白蛋白偶联,免疫雌性Balb/c系小鼠;利用单克隆抗体技术完成杂交瘤融合,并用非竞争及竞争ELISA法,对阳性克隆进行鉴定。杂交瘤诱生腹水后,经亲和纯化,用于15 nm胶体金标记,制作玉米赤霉醇胶体金检测试纸。融合细胞经筛选,最终对3株杂交瘤细胞建株,结果均为Ig G1型。其中Mab-1A8诱生腹水效价高达1∶1.28×10^7,对玉米赤霉醇抑制中浓度（I50）为10.25 ng·m L^-1,检测灵敏度（I10）为1.96 ng·m L^-1,线性检测范围在2.96-35.46 ng·m L^-1,对同系物玉米赤霉烯酮的交叉反应率小于0.1%。试验研制的玉米赤霉醇胶体金试纸,检测阈值为2.5μg·m L-1,检测时间为10 min。本研究制备了一种新型玉米赤霉醇特异性单克隆抗体,并研制了以单抗为基础的玉米赤霉醇胶体金快速检测试纸,为玉米赤霉醇残留快速检测提供了灵敏、便捷的新方法。In previous study,monoclonal antibodies against zeranol showed high cross-reactivity to zearalenone,which is a common mycotoxin produced by Fusarium spp. Three female BALB / c mice were immunized with the conjugate of zeranol-16-carboxypropylether and bovine serum album. The hybridoma cells were produced by the monoclonal antibodies technology. And non-competitive and competitive ELISA was employed to detect positive clones. A lateral flow immunoassay has been established based on conjugate of purified monoclonal antibodies with 15 nm colloidal gold.The isotypes of all three hybridoma cells were identified as Ig G1 type. The titer of Mab-1A8 ascites fluid was 1∶ 1. 28× 10^7,and after purification by Protein G,the I50 of purified antibodies was 10. 25ng·m L^-1. The linear range for zeranol was between 2. 96 to 35. 46ng·m L^-1and the detection limit（ I10） was 1. 96ng·m L^-1. Cross reactivity of the antibodies with zearalenone was lower than 0. 1%. The cut-off concentration of zeranol for the lateral flow assay was2. 5μg·m L-1with 10 minutes detection time. Novel monoclonal antibodies with improved specificity and colloidal gold rapid test strips have been produced for zeranol. This article provides a sensitive and convenient detection method for zeranol residues.

关 键 词：玉米赤霉醇 单克隆抗体 玉米赤霉醇-16-羧丙基醚 玉米赤霉烯酮 胶体金检测试纸 

分 类 号：S859.84[农业科学—临床兽医学]