强力霉素单克隆抗体制备及ciELISA检测方法的建立  

Preparation of monoclonal antibody and development of ciELISA method for rapid detection of doxycycline

作　　者：余少梅[1,2] 艾晓辉[2] 甘金华[2] 刘永涛[2] 索纹纹[1,2] 

机构地区：[1]华中农业大学水产学院,武汉430070 [2]中国水产科学研究院长江水产研究所,武汉430223

出　　处：《华中农业大学学报》2013年第3期112-117,共6页Journal of Huazhong Agricultural University

基　　金："十一五"国家科技支撑计划"渔药安全使用及快速检测技术研究"项目(2006BAD03B04-04);中央公益性研究院所基本科研业务费专项资金项目(2011JBFA19)

摘　　要：以人工合成的强力霉素-牛血清白蛋白(DC-BSA)为抗原免疫BALB/C小鼠,用间接ELISA和间接竞争ELISA法选择细胞融合的备用小鼠;应用杂交瘤技术建立分泌DC McAb的杂交瘤细胞株,用体内诱生法制备DC McAb,并对DC McAb的免疫学特性进行鉴定;应用DC McAb建立检测DC的ciELISA方法,并对其相关特性进行检测。结果表明:筛选后获得1株分泌特异性抗体的细胞3D1-H4,细胞培养上清效价为1∶(8×102),腹水效价为1∶(5.12×105),抗体为IgG1型免疫球蛋白,与四环素、土霉素和金霉素分别有8.87%、5.86%和2.74%的交叉反应率,与其他抑制物无交叉反应性;ciELISA检测方法的线性范围为1.58～199.53 ng/mL,灵敏度为2.88 ng/mL,半数抑制的质量浓度IC50为36.31 ng/mL,斑点叉尾鮰肌肉和肝脏样品中DC的平均添加回收率分别为85.13%和79.69%,平均批间和批内变异系数均小于15%,不同基质对检测结果影响小。所建立的ciELISA方法可以应用于水产品中DC残留的检测。To detect doxycycline （D C） residues in aquatic products,a ciELISA method were developed through preparing monoclonal antibody （McAb） by immunizing BALB/C mice, using doxycylinebovine serum albumin conjugate （DC-BSA） as immunogen. The BALB/C mice used in the experiment for cell fusion were selected by two methods, the indirect ELISA and the indirect competitive ELISA. A hybridoma cell strain （3D1-H4）, which can secret a McAb that specifically reacted with DC, were ob- tained. The McAb was separated by using hybridoma technology,and it was identified to belong to IgG1 by the indirect ELISA. A ciELISA for detecting DC was then developed, using this McAb. This McAb was characterized on its sensitivity, IC50 and veracity. The results showed that the titers of the McAb were 1 ： （8×10^2） and 1 ： （5.12×10^5） in supernatant and ascites,respectively. This McAb had 8.87%, 5.86 % and 2. 74% cross-reactivity with tetracycline, oxytetracycline and chlotetracycline, respectively. The linear detection range for this ciELISA varied from 1.58 to 199. 53 ng/mL, with a sensitivity of 2.88 ng/mL and an IC50 of 36.31 ng/mL. The recoveries of DC spiked in fish muscle and liver were 85.13% and 79. 69%, respectively. Both the inter-assay and intra-assay coefficient variations were less than 15%. These results suggested that this ciELISA method was able to be used for detecting DC residues in aquatic products.

关 键 词：强力霉素(DC) 杂交瘤细胞 单克隆抗体 间接竞争酶联免疫吸附法(ciELISA) 水产品 残留检测 

分 类 号：S948[农业科学—水产养殖]