基于氯霉素单克隆抗体的ELISA方法的建立  

Study on An Enzyme Linked Immunosorbent Assay Based on A Monoclonal Antibody for the Detection of Chloramphenicol

作　　者：张立辰[1] 生威[1] 胡高爽 张燕[1] 王硕[1] 

机构地区：[1]天津科技大学食品工程与生物技术学院教育部食品营养与安全重点实验室,天津300457

出　　处：《食品研究与开发》2015年第24期127-130,共4页Food Research and Development

基　　金："十二五"国家科技支撑计划(2012BAD28B05);天津市科技计划项目(11ZCGHHZ01100)

摘　　要：旨在建立一种用于检测海产品中氯霉素残留的ELISA检测方法。利用活化酯法将氯霉素半抗原连接到载体蛋白KLH上,制备氯霉素人工抗原,并免疫小鼠。通过杂交瘤技术筛选出一株能稳定分泌抗体的细胞株10A3B6E,经小鼠体内培养制备腹水并经纯化得到单克隆抗体。单克隆抗体的重链为Ig G1,轻链为Kappa。通过对免疫分析方法工作条件的优化,建立了一种简便、快捷、实用的检测氯霉素的直接竞争ELISA方法。该方法 IC50值为（0.54±0.04）ng/m L,IC15值为（0.10±0.03）ng/m L。该抗体与氯霉素琥珀酸钠、甲砜霉素、氟甲砜霉素、链霉素的交叉反应率分别为78%、0.09%、0.25%和0.05%,与四环素的交叉反应率低于0.002%,说明该抗体是一种高特异性抗体。利用所建立的直接竞争ELISA方法对鳕鱼和虾样品进行检测,添加回收率在62.6%~120.0%之间,变异系数在0.94%~13.52%之间。本研究所建立的直接竞争ELISA方法具有较好的准确性和灵敏度,可以用来对海产品中的氯霉素残留情况进行检测。The aim of this research was to develop an ELISA to detect chloramphenicol in marine products. The chloramphenicol succinate was conjugated to keyhole limpet hemocyanin by activated ester method to prepare the immunogen for immunizing mices. Through a hybridoma cell line, a strain of cells which can produce antichloramphenicol monoclonal antibody was obtained. Ascites was produced by mice vivo culcure and purified to obtain monoclonal antibody. The heavy chain of the antibody was IgG1, and the light chain was Kappa. A direct competitive ELISA using above-mentioned monoclonal antibody was developed for the detection of chloramphenicol by optimizing the working conditions of assay. The IC（50） value was（0.54 ±0.04） ng/m L, IC15 value was（0.10 ±0.03） ng/m L. Cross-reactivities with chloramphenicol sodium succinate, thiamphenicol,florfenicol, streptomycin and tetracycline were 78 %, 0.09 %, 0.25 %, 0.05 % and 0.002 % respectively,which indicated that the assay had high specificity. The recoveries for chloramphenicol from cod and shrimp samples were ranged from 62.6 % to 120 % and the coefficients of variation were between 0.94 %-13.52 %. The ELISA allows for a rapid, sensitive, specific, accurate, and low-cost determination of chloramphenicol residues in marine products.

关 键 词：氯霉素 海产品 兽药残留 单克隆抗体 直接竞争ELISA 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]