摘要
目的 :建立敏感、特异和快速的针对T 2毒素的ELISA检测方法 ,形成具有我国自主知识产权的快速检测试剂盒。方法 :利用B细胞杂交瘤技术 ,建立抗T 2毒素的单克隆杂交瘤细胞株。结果 :用T 2 羊免疫球蛋白 (T 2 GIgG)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系Sp2 /0融合 ,经过 3～ 4次亚克隆建立了 2个稳定分泌抗T 2毒素抗体的杂交瘤细胞株 ,分别命名为 2A7和 6E4。将上述 2株杂交瘤细胞分别注入BALB/c小鼠腹腔 ,获得含抗T 2毒素单克隆抗体的腹水 ,并将腹水用饱和硫酸铵法进行纯化 ,得到 2A7和 6E4单克隆抗体。两株单克隆抗体的Ig亚类为IgG1,腹水中抗体的滴度分别为 6 .4× 10 6和 1.1× 10 7,参考工作浓度分别为1.0× 10 6和 3.2× 10 6。纯化后 2A7抗体的IgG含量为 16 .4g/L ,亲和常数为 8× 10 -8mol/L。 2A7抗体与其他结构类似物无交叉反应 ,具有较高的特异性。结论 :制备了具有高特异性和亲和力的抗T 2毒素单克隆抗体 ,初步建立了针对T 2毒素的ELISA检测方法。
Objectives: To develop a sensitive, specific and rapid enzyme-linked immunosorbent assay for detection of T-2 toxin. To develop rapid detection kit that possesses patent of our country. Methods: Hybridoma cell line excreting monoclonal antibodies against T-2 toxin were produced using B cell hybridoma technique. Results: Two hybridoma cell lines secreting monoclonal antibodies against T-2 toxin, coded 2A7 and 6E4 respectively, were obtained by fusing murine Sp2/0 cells with spleen cells from BALB/c mice immunized with T-2-GIgG conjugate and subcloning for 3 to 4 cycles. The ascites containing monoclonal antibodies against T-2 toxin was gained via inoculation of the hybridoma cell into abdominal cavity of BALB/c mice. Monoclonal antibodies produced by the hybridoma cells were tested for subtypes and designated as IgG1 for both 2A7 and 6E4. The titers of antibodies in ascites were 6.4×106 and 1.1×107 and the reference working concentrations were 1.0×106 and 3.2×106 respectively. IgG concentration in purified ascites yielded by hybridoma cell with the code number of 2A7 reached 16.4g/L and its affinity constant was 8×10 -8 mol/L. The monoclonal antibodies obtained in the present study was highly specific to T-2 toxin, because no cross reactions between the monoclonal antibodies against T-2 toxin and against the analogues of T-2 toxin were found. Conclusion: Monoclonal antibodies against T-2 toxin that have high specificity and affinity were produced and an ELISA detection method for T-2 toxin was established. [
出处
《军事医学科学院院刊》
CSCD
北大核心
2004年第4期311-313,318,共4页
Bulletin of the Academy of Military Medical Sciences
基金
"十五"国家科技重大专项攻关项目( 2 0 0 1BA80 4A2 0 )
