牛奶中玉米赤霉烯酮和氯霉素二合一检测芯片的开发研究  

Development of immunochips for simultaneous detection of zearalenone (ZEN) and chloramphenicol (CAP) in milk

作　　者：李丽[1] 张晓龙 任帅 丁乔棋 宋素泉[1] 闫丽萍 LI Li;ZHANG Xiaolong;REN Shuai;DING Qiaoqi;SONG Suquan;YAN Liping(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Heilongjiang Agricutural Information Center,Harbin 150008,China;Jiangsu Engineering Laboratory of Animal Immunology,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Detection Center of Terrestrial Wildlife Disease,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,南京210095 [2]黑龙江省农业信息中心,哈尔滨150090 [3]南京农业大学江苏省动物免疫工程实验室,南京210095 [4]江苏省陆生野生动物疫源疫病检测中心,南京210095

出　　处：《黑龙江畜牧兽医》2019年第3期15-19,23,175,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0501009;2016YFD0501200);中央高校基本科研业务费专项资金项目(Y0201500195);江苏省自然科学基金面上项目(BK20161452);广东省科技计划重点项目(2017B020202010);江苏高校优势学科建设工程项目

摘　　要：为了开发出能同时检测玉米赤霉烯酮(ZEN)和氯霉素(CAP)的二合一免疫芯片,试验首先制备抗ZEN的单克隆抗体,并结合抗CAP多克隆抗体采用间接竞争ELISA方法进行芯片组装,最终确定定量标准曲线,并进行加标样品的检测。结果表明:共筛选得到3株抗ZEN的单克隆抗体(1C5、2B10、4F3),选择其中的1株杂交瘤细胞1C5制备小鼠腹水,抗体效价为5.12×10~4,半抑制浓度(IC_(50))为6.57 ng/mL。试验建立的免疫芯片对ZEN和CAP的最低检测限分别为2.84 ng/mL、9.49 ng/mL。在牛奶加标回收试验中,ZEN回收率为95.2%～109.8%,CAP回收率为95.1%～100.3%,回收率均较高,且与高效液相色谱(HPLC)法检测结果有良好的相关性,变异系数(CV)小于15.0%。说明该芯片能用于检测实际样品中的ZEN和CAP。The aim of the present study was to develop an immunochip for simultaneous detection of zearalenone ( ZEN) and chloramphenicol ( CAP) . Anti-ZEN monoclonal antibody and anti-CAP polyclonal antibody were prepared firstly. Then immunochip was assembled based on indirect competition ELISA. Finally,quantitative standard curves were established,and the labeled samples were detected. The results showed that three strains of stable hybridoma clones ( 1C5,2B10,4F3) against ZEN were obtained. One strain of the hybridoma cells,1C5,was selected to prepare ascites of mice. The titer of antibody was 5. 12×104,and the half inhibitory concentration ( IC50 ) was 6. 57 ng /mL. The detection limits of ZEN and CAP were 2. 84 ng /mL and 9. 49 ng /mL,respectively. The recovery rate of the milk was good in the recovery test of adding standard. The recoveries of ZEN and CAP were 95. 2%- 109. 8% and 95. 1%- 100. 3%,respectively. It had a good correlation with HPLC,and the coefficient of variation ( CV) was lower than 15. 0%. The results indicated that the chip could be used to detect the ZEN and CAP of real samples.

关 键 词：玉米赤霉烯酮 氯霉素 免疫芯片 单克隆抗体 间接竞争ELISA法 化学发光 

分 类 号：S859.83[农业科学—临床兽医学]