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目的制备可高效、特异性富集肠出血性E.coli O157(enterohemorrhagic Escherichia coli O157,EHEC O157)的免疫磁珠。方法分别用5种不同规格磁珠与E.coli O157特异性单克隆抗体偶联制备免疫磁珠,通过比较磁珠蛋白偶联量,选取包被效果最佳的磁珠规格;对制备工艺进行优化(包括活化时间、温度,偶联缓冲液p H值,偶联温度、时间);对制备的O157免疫磁珠选择性集菌的敏感性和特异性进行评价,并与进口磁珠吸附率进行比较。结果直径0.5~1.0μm羧基磁珠抗体包被效果及目的菌分离效果最好。加入NHS后,活化30 min为最佳时间;4℃磁珠活化温度、0.01 mol/L PBS(p H 7.4)构成的体系对磁珠偶联单克隆抗体效果最佳;偶联温度为37℃、偶联时间为120 min时,偶联蛋白量最高,且波动范围较小,为最佳偶联温度及时间。在复杂的微生物环境中,O157免疫磁珠的敏感性达到20 CFU/ml。在混合菌液中,E.coli O157菌含量在1.4×10~3 CFU/ml时,免疫磁珠特异性捕获率达到90%。结论获得羧基磁珠与E.coli O157单克隆抗体偶联的最佳条件。制备的免疫磁珠能够特异性富集EHEC O157,具有操作简便,分离速度快,捕获率高等特点,可用于临床样品或食品中EHEC O157的分离鉴定。
Objective To prepare immunomagnetic beads that can efficiently and specifically enrich enterohemorrhagic Escherichia coli O157 (EHEC O157). Methods The magnetic beads were prepared by coupling five kinds of magnetic beads of different specifications with the specific monoclonal antibody of E. coli O157 respectively. By comparing the amount of magnetic bead coupling, the optimal bead size was selected. The preparation process (Including activation time, temperature, p H value of coupling buffer, coupling temperature and time). The sensitivities and specificities of the prepared O157 immunomagnetic beads for selective bacterial collection were evaluated and compared with those of imported magnetic beads Compare. Results The diameter of 0.5 ~ 1.0μm carboxyl beads antibody coating effect and the purpose of separation of bacteria best. After adding NHS, activation for 30 min was the best time. The optimal conditions were as follows: the activation temperature of the magnetic beads at 4 ℃, the system composed of 0.01 mol / L PBS (p H 7.4) , Coupling time is 120 min, the highest amount of coupling protein, and the fluctuation range is smaller, the optimal coupling temperature and time. The sensitivity of O157 immunomagnetic beads to 20 CFU / ml in a complex microbiological environment. In the mixture of bacteria, E. coli O157 bacteria content of 1.4 × 10 ~ 3 CFU / ml, the immune magnetic beads specific capture rate of 90%. Conclusion The optimal conditions for the conjugation of carboxyl magnetic beads with monoclonal antibodies against E. coli O157 were obtained. The prepared immunomagnetic beads can specifically enrich EHEC O157, which has the advantages of simple operation, fast separation and high capture rate, and can be used for the separation and identification of EHEC O157 in clinical samples or foods.