高特异性抗桔霉素单克隆抗体的制备及鉴定

Preparation and Characterization of The High Specificity Monoclonal Antibodies Against Citrinin

以1,4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白偶联抗原CIT-BSA,将偶联抗原免疫BALB/C小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株稳定分泌抗桔霉素抗体的杂交瘤细胞株H2-F8.该单克隆抗体经过初步鉴定,抗体类型为IgM类,抗体的相对亲和力为4.17×108L/mol,单抗与黄曲霉毒素B1、赭曲霉毒素A、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等毒素的交叉反应率均低于0.1%,与红曲色素中的橙色素和红色素的交叉反应率均低于0.01%.在此基础上建立了间接竞争ELISA检测方法,线性范围为0.05～1.0μg/L,IC50值为0.3μg/L.结果为快速检测桔霉素的酶联免疫检测方法的建立和检测试剂盒的研制提供技术依据.

To prepare citrinin（CIT） -protein antigen,CIT was conjugated with bovine serum albumin（BSA） by 1,4-butanediol diglycidyl ether.The spleen from the BALB/C mice immunized with CIT-BSA conjugate was used to fuse with the murine SP2/0.By subcloning,a hybridoma cell lines excreting monoclonal antibodies（McAb） against CIT was obtained and named H2-F8.The monoclonal antibodies obtained from hybridoma H2-F8 was of IgM subclass.The affinity constant of the McAb to CIT was 4.17×10^8 L/mol and the IC50 value was 0.3μg/L.Its linear range of the assay was between 0.05μg/L and 1.0μg/L.The cross-reactivity rates were less than 0.1%of other toxins such as ochratoxin A,aflatoxin B1,deoxynivalenol,zearalenone and were less than 0.01%of rubropunctatine and rubropunctamine.This work would be helpful for establishing the technology and developing the kit to determine CIT-contaminated samples by ELISA.