将骨髓瘤细胞SP2/0与出血性大肠杆菌O157∶H7免疫小鼠得到的脾细胞融合,通过筛选得到3株稳定分泌抗出血性大肠杆菌O157∶H7单克隆抗体的杂交瘤细胞株D3、E7、B9,D3和B9亚类为IgG1,E7亚类为IgG2a,轻链亚型均为κ。交叉反应结果显示这3株单抗仅结合出血性大肠杆菌O157∶H7,对15株其他细菌无反应,测得胶体金标记单抗最佳pH、最佳结合量分别为D3:pH 7.5~8.0、24μg/mL;B9:pH 7.5、12μg/mL;E7∶pH7.5、18μg/mL,抗体配对选择胶体金结合D3喷涂金标垫、E7以1 mg/mL喷涂NC膜作T线为最优组合,试纸条检测出血性大肠杆菌O157∶H7灵敏度为2.1×106CFU/mL,且仅可检出出血性大肠杆菌O157∶H7,对25株其他细菌均无交叉反应,模拟带菌实验表明,对市售面包、牛奶、果冻各25 g(mL)均添加约200 CFU出血性大肠杆菌O157∶H7,增菌培养8、10、10 h即可检出。研究表明,实验自主制备的抗体性能良好,试纸条特异性、灵敏度达到国外同类产品,可在政府食源性致病菌监管部门、食品企业推广运用。

Fusing the Myeloma cells SP2 /0 was fused with spleen from Escherichia coli O157∶ H7 immune mice.Three hybridoma cell lines( D3,E7 and B9) secreting anti EHEC O157∶ H7 monoclonal antibody were obtained after screening,wherein the subtype of D3 and B9 belonged to IgG1,the subtype of E7 belonged to IgG2a,and their light chain subtypes all were κ. The cross reaction results showed that these 3 lines of monoclonal antibody didn't respond to the other 15 bacteria strain except the EHEC O157∶ H7. The best pH for monoclonal antibody labeled with Colloidal gold and their binding capacity were as follows: pH7. 5 ~ 8. 0 and 24 μg /mL for D3,pH7. 5 and 12 μg /mL for B,pH7. 5 and 18 μg /mL for E7. The selected best antibody pairs were the optimal combination of gold pad sprayed with colloidal gold-labelledD3 and NC film coated with 1 mg /mL E7 as T line. The sensitivity of test strip for detection of EHEC O157∶ H7 was 2. 1 × 106CFU /mL. The other 25 bacteria strains had no cross-reactivity. The results from bacteria simulation experiments showed that EHEC O157∶ H7 was detectable in commercially available bread,milk and jelly after 200 CFU EHEC O157∶ H7 was added into 25 g /mL each of them and enrichment cultured for 8 h,10 h and10 h. This study showed that the independent preparated antibody has good performance,and the specificity and sensitivity of test trip were similar as those of foreign products,thus it could be widely used in government food-borne pathogen regulatory authorities and food enterprises.