跗关节免疫法抗镉单克隆抗体的制备

Production of two anti-cadmium monoclonal antibodies by hock immunization

为快速方便地检测环境水样中的镉离子浓度,以Cd-ITCBE-KLH为免疫抗原,采用小鼠跗关节注射抗原法,分2次免疫BALB/c小鼠,每次注射20μg抗原,首次免疫后第20天收获皮下腘淋巴结,采用常规方法与骨髓瘤细胞融合,筛选细胞株,以体内诱生腹水法收获单克隆抗体,纯化后建立了镉离子的竞争ELISA检测方法。结果显示,共筛选了A2G11、A4B8B1C2、B1G5、B4F12五株杂交瘤细胞,采用其中的1株A2G11细胞株建立了镉离子的竞争ELISA检测方法,其工作曲线方程为y=-0.119 5lg[Cd2+]+0.615 4,r2=0.973 2;半数抑制浓度为1.485μg/L,检测范围(IC20～IC80)为0.029～762.676μg/L,最低检测限为0.102μg/L;与其他金属离子的交叉反应≤3.684%。表明用该方法能够有效制备抗金属离子的单克隆抗体。

In order to establish a rapid and simple method for the determination of concentration of cadmium ions（Cd2＋） in environmental water samples,20 μg Cd-ITCBE-KLH was used as a complete antigen and injected into BALB/c mice＇s hock by subcutaneous injection.The procedure was repeated 2 weeks later.The popliteal lymph nodes were harvested after 20 days of the priming.The traditional fusing and screening methods were used to generate hybridoma cells.Then ascites were induced for harvesting monoclonal antibody in vivo.After purified,the monoclonal antibody was used to establish a competitive ELISA for the detection of Cd2＋.Five hybridoma cell lines named A2G11,A4B8,B1C2,B1G5 and B4F12 were generated and the A2G11 was employed to establish a competitive ELISA with the equation of y=-0.119 5 lg＋0.615 4,r2= 0.973 2.The IC50 and the detection limit of the assay was 1.485 μg/L and 0.102 μg/L respectively,and the detection range（IC20 to IC80） was 0.029 to 762.676 μg/L.The cross reactivity with other metal ions was less than 3.684%.All the results indicated that hock immunization was able to generate monoclonal antibody.