抗氯霉素单克隆抗体的制备及直接竞争ELISA方法的建立  

Preparation of monoclonal antibody against chloramphenicol and establishment of direct competitive ELISA for determination of chloramphenicol

作　　者：陈正贤[1] 施曼玲[2] 李因来[2] 叶兴乾[3] 沈立荣[3] 

机构地区：[1]浙江大学农业与生物技术学院生物技术研究所,浙江杭州310029 [2]杭州师范大学生命与环境科学学院,浙江杭州310036 [3]浙江大学生物系统工程与食品科学学院食品科学与营养系,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2010年第2期133-138,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：杭州市科技局市属高校重点实验室科技创新资助项目(2006831H07);浙江省科技计划资助项目(2004C32052)

摘　　要：用人工合成的氯霉素-卵清蛋白（CAP—OVA）免疫BALB／c小鼠，通过杂交瘤技术筛选出1株能稳定传代并分泌抗氯霉素（CAP）单克隆抗体（McAb）的杂交瘤细胞4C9，并以此制备腹水单抗．经间接竞争ELISA（ciELISA）检测，该单抗亚类重链类型是IgG1，轻链为K类型，单抗腹水效价为1：1×10^6，与甲砜霉素、氟甲砜霉素以及其他常见抗生素的交叉反应率小于0．01％．用过碘酸钠氧化法合成CAP酶标记单抗，建立了CAP直接竞争ELISA（cdELISA）方法．此法检测的线性范围为0．1～100ng·mL^-1，半数抑制浓度（IC50）为5．81ng·L^-1添加回收实验表明所建立的CAPcdELISA方法检测限达到0．1ng·L^-1，对比试验表明其检测灵敏度与商品试剂CAP ELISA基本相当．Through immunization of BALB/c mice with synthesized CAP-ovalbumin （CAP-OVA）, a hybridoma cell line, the 4C9 which eould produce monoclonal antibody against chloramphenicol （CAP） and propagate stably, was established. With the myeloma cells, the ascites containing monclonal antibody （McAb） were prepared. The indirect competitive ELISA （ciELISA） test revealed the heavy chain and light chain of McAb were IgG1 and κ, respectively. The ELISA titer of ascites was 1：1×10^6 . The percentages of crossactivity to thiamphenicol, florfenicol and other antibiotics were all less than 0. 01%. A horseradish peroxidase （HRP）-labeled antibody was synthesized by the sodium periodate reaction, then a direct competitive enzymelinked immunosorbent assay （cdELISA） was developed. Quantization of the CAP was linear from 0. 1 to 100 ng-mL 1, and the hemi-inhibitory concentration （ICs0） was 5.81 ng·L^-1. The recovery test showed that the detection limit for CAP was 0. 1 ng·L^-1 in cdELISA. Comparative test showed that detected sensibility of the monoelonal antibody was nearly equivalent to the commercial CAP ELISA kit.

关 键 词：氯霉素 单克隆抗体 酶标抗体 直接竞争ELISA 

分 类 号：S852.43[农业科学—基础兽医学] Q813[农业科学—兽医学]