抗玉米赤霉烯酮单克隆抗体的制备

Development of monoclonal antibodies against zearalenone

为建立快速、简单、灵敏、有效的玉米赤霉烯酮的检测方法,将玉米赤霉烯酮与羧甲氧基胺半盐酸盐反应,合成半抗原ZEN-oxime,通过活泼酯法将半抗原与载体蛋白偶联制备玉米赤霉烯酮人工抗原,以人工抗原ZENBSA免疫BALB/C小鼠,取该鼠脾细胞与SP 2/0鼠骨髓瘤细胞融合,经筛选和克隆,得到了1株能稳定分泌抗玉米赤霉烯酮抗体的单克隆细胞株3D10。3D10的抗体类型及亚类均为IgM,其轻链为Kappa链。制备的单克隆抗体腹水通过间接酶联免疫吸附测定,效价在1×10-6以上。该单克隆抗体对玉米赤霉烯酮的IC50为225 ng/ml,除与玉米赤霉烯酮结构类似物有一定的交叉反应外,与其他参试真菌毒素均无交叉反应。所制备的单克隆抗体可用于玉米赤霉烯酮毒素初筛和定性检测。

To develop a rapid,simple, sensitive and effective method for detecting zearalenone（ZEN）, the haptenZEN-oxime were synthesized through the reaction between zearalenone and carboxymethoxylamine hemihydrochloride. Thehapten was conjugated to carrier protein to form the complete antigens by NHS ester method. One hybridoma cell line （3D10）secreting monoclonal antibody （McAb） against ZEN was produced by fusing mouse myeloma cells （SP 2/0） with spleen cellsof BALB/ C mice immunized by artificial antigen conjugated with bovine serum albumin（BSA）. Isotype and subclass of themonoclonal cell line （3D10） belonged to IgM. The light chain of the McAb was identified as Kappa. The titre of ascitic fluidswas up to 1 伊l0-6 by indirect ELISA. There were crossreactions between the monoclonal antibodies and ZEN andstructural analogues. There was no cross reaction betweenZEN monoclonal antibodies and the other three mycotoxins.The IC50 of McAb against ZEN was 225 ng/ ml. In conclu-sion, the McAb prepared can be used for preliminary andqualitative detection of ZEN residue.