阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

Preparation of Monoclonal Antibody against Enterobacter sakazakii and Establishment of an ELISA Detection Method

试验旨在制备抗阪崎肠杆菌的单克隆抗体,初步建立其ELISA检测方法。以灭活的阪崎肠杆菌全菌体为抗原免疫BALB/c小鼠,筛选血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,制备杂交瘤细胞,并用间接ELISA法选取阳性杂交瘤细胞,扩大培养后测定单克隆抗体的效价,进行特异性及抗体间的配对,使用mAb亚类检测试剂盒鉴定单克隆抗体的亚型,并利用得到的抗体建立双抗体夹心ELISA检测方法。本试验得到3株具有良好特异性能稳定分泌单克隆抗体的阳性细胞株5C10、2B6和Ab02,经两两配对,据阳性D450nm值及P/N值选择1∶20000稀释的5C10作为包被抗体,1∶40000稀释的Ab02作为酶标二抗,建立ELISA检测法,应用建立的方法与荧光定量PCR方法检测动物实验室保存的20份进出口送检奶粉样品,结果显示试验结果一致。本试验成功制备阪崎肠杆菌的单克隆抗体并建立其ELISA检测法,为大批量快速检测阪崎杆菌奠定了基础。

The assay was aimed to prepare the monoclonal antibody against Enterobacter sakazakii and establish the ELISA detection method. The splenocytes from immunized mice were fused with myeloma cells SP2/0, and hybridoma cells were screened by indirect ELISA.Then culturing more monoclonal antibodies, ascites titers were identified with indirect ELISA.Subclass was determinated by mouse isotype. Double-antibody sandwich ELISA detection method was established by using the antibodies. Three hybridoma cell strains that could secret monoclonal antibodies against Enterobacter sakazakii stably were obtained,named as 5C10, 2B6 and Ab02, paired 5C10 as coating antibody and Ab02 as HRP-secondary antibody, to establish ELISA detection method. We compared with fluorescence PCR method to test 20 portion imports of milk powder, ELISA detection method had the same consequence. The monoclonal antibody against Enterobacter sakazakii and ELISA detection method had been successfully established, which laid the foundation for rapid detection of Enterobacter sakazakii in larger number.