阪崎肠杆菌单克隆抗体的制备与特性鉴定

目的:制备抗阪崎肠杆菌的单克隆抗体并对其生物学特性进行鉴定。方法:采用灭活的阪崎肠杆菌菌体为抗原,免疫BALB/c小鼠,取血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,mAb亚类检测试剂盒鉴定单克隆抗体的亚型,通过Western blot和间接ELISA法鉴定该单克隆抗体的特性、效价及mAb相对亲和力。结果:获得2株能稳定分泌抗阪崎肠杆菌mAb的杂交瘤细胞株,分别命名为1H7、2B12,抗体Ig亚类分别为IgG1和IgG2b;交叉反应显示单抗具有良好的特异性;ELISA分析表明制备的单抗效价在1×107～2×107,相对亲和常数达109L/mol,染色体鉴定分别为104和106条,符合杂交瘤细胞的特性。结论:抗阪崎肠杆菌单克隆抗体的成功制备为其快速检测方法的建立奠定了基础。

Production and characterization of the monoclonal antibodies against Enterobacter Sakazakii

Objective:To prepare the monoclonal antibody(mAb) against Enterobacter Sakazakii and characterize their biological functions.Methods:Female BALB/c mice were immunized with Enterobacter Sakazakii inactivated.The splenocytes from immunized mice were fused with myeloma cells SP2/0,and hybridoma cells were screened by indirect ELISA.Subclass was determinated by mouse isotype.The specifities of mAbs,ascites titer,supernatant titer and relative affinities of mAbs were identified with Western blot and indirect ELISA.Results:Two hybridoma cell strains that could secret Enterobacter Sakazakii stably were obtained,named as 1H7 and 2B12,appraisal to its subclass,respectively was IgG1 and IgG2b;The cross reactivity demonstration,two mAbs were specific to Enterobacter Sakazakii.The titers of obtained mAbs were 1×107-2×107,and affinity constant was 109L/mol;the chromosome appraisal,respectively was 104 and 106 chromosomes,conforms to the hybridoma cell’s characteristic.Conclusion:The monoclonal antibody against Enterobacter Sakazakii has been successfully obtained,which established foundations for the related detection method of Enterobacter Sakazakii.