抗玉米赤霉烯酮单克隆杂交瘤细胞系的建立及单抗生产

Development of hybridoma cell lines excreting monoclonal antibodies against zearalenone

为快速检测食品中的玉米赤霉烯酮 (Zearalenone以下简称ZEN) ,建立了抗ZEN的单克隆杂交瘤细胞株。用ZEN BSA偶联物免疫 8～ 10周龄雌性BalB c小鼠后 ,取脾细胞与小鼠骨髓瘤细胞Sp2 0融合 ,经过 4～ 5次亚克隆建立了稳定分泌抗ZEN抗体的杂交瘤细胞株 ,分别命名为Z2A4、Z8D6。将上述 2种杂交瘤细胞分别注入BalB c小鼠腹腔 ,获得含抗ZEN单克隆抗体的腹水。将腹水用饱和硫酸铵盐析法纯化 ,得到Z2A4、Z8D6单克隆抗体。经鉴定 ,其亚类为IgG1,抗体腹水效价Z2A4和Z8D6分别为 1∶1 6× 10 6和 1∶3 2× 10 6;分子量均为 15 0 0 4 0 (15 0kD) ;参考工作浓度分别为1∶4 0 0 0 0和 1∶10 0 0 0 0 ;纯化后抗体IgG含量分别为 34和 39mg ml ;抗体与其它霉菌毒素无交叉反应(交叉反应率 <1% ) ,具有较高特异性 ;抗体亲和力常数Z2A4和Z8D6分别为 5 5 2× 10 8和 4 6 8×10 9mol L。

Two hybridoma cell lines excreting monoclonal antibodies against Zearalenone (ZEN), coded Z2A4 and Z8D6, respectively, were obtained by fusing murine Sp2/0 cells with spleen cells from 8～10-week-old BalB/c mice immunized with ZEN-BSA conjugate and subcloning for 4 to 5 cycles .The ascites containing monoclonal antibodies against ZEN was gained via inoculation of the hybridoma cells into the abdominal cavity of BalB/c mice. The monoclonal antibodies produced by the hybridoma cells were tested for subtypes and designated as IgG 1 for ZEN. The titer of antibodies in ascites was 1∶ 1.6×106 for Z2A4 and 1∶3.2×106 for Z8D6, the MW was both 150 040(150 kD) and the working concentration was 1∶40 000 and 1∶100 000. IgG concentration in purified ascites yielded by hybridoma cells of Z2A4 and Z8D6 reached 34 and 39 mg/ml respectively, and had high specificity to ZEN because there was no cross reaction between the monoclonal antibodies against ZEN and other mycotoxins. Affinity constants of Z2A4 and Z8D6 were 5.52×108 and 4.68×109 mol/L respectively.