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目的:制备用于特异性分离阪崎肠杆菌的免疫磁性壳聚糖微球以及对阪崎肠杆菌的捕获效果。方法:采用反向悬浮交联法制备磁性壳聚糖微球,并对微球的表面进行氨基化修饰,然后与阪崎肠杆菌多克隆抗体进行偶联,制备免疫磁性壳聚糖微球。优化磁性壳聚糖微球对阪崎肠杆菌多克隆抗体的偶联条件,包括偶联时间、磁性壳聚糖微球用量、偶联体系p H,对抗体的饱和偶联量。通过与显色培养基结合的方法研究免疫磁性壳聚糖微球对阪崎肠杆菌的捕获能力。结果:制备的磁性壳聚糖微球通过表面修饰能连接上大量的活性氨基,在p H 7.4的偶联体系中,10.0 min即可完成对阪崎肠杆菌多克隆抗体的偶联,0.01 g磁性壳聚糖微球对抗体的饱和偶联量为25~50μL。当阪崎肠杆菌浓度较低时,制备的免疫磁性壳聚糖微球对阪崎肠杆菌的捕获率达94.7%,对阪崎肠杆菌的灵敏度为5 cfu/m L。结论:获得阪崎肠杆菌免疫磁性壳聚糖微球,该微球是一种非常有潜力的快速富集、分离阪崎肠杆菌的有效方法。
OBJECTIVE: To prepare immunomagnetic chitosan microspheres for specific isolation of Enterobacter sakazakii and its capture effect on Enterobacter sakazakii. Methods: Magnetic chitosan microspheres were prepared by reverse levitation cross-linking method, and the surface of the microspheres was modified by amination. Then the immunomagnetic chitosan microspheres were prepared by coupling with the polyclonal antibody of Enterobacter sakazakii. Optimization of the coupling conditions of magnetic chitosan microspheres to E. sakazakii polyclonal antibody, including the coupling time, the amount of magnetic chitosan microspheres, the coupling system p H, the saturation coupling amount of the antibody. The binding ability of immunomagnetic chitosan microspheres to Enterobacter sakazakii was studied by combining with chromogenic medium. Results: The prepared magnetic chitosan microspheres could be linked to a large amount of active amino groups by surface modification. The conjugation of polyclonal antibodies to Enterobacter sakazakii was completed in 10.0 μL in the coupling system of p H 7.4, and 0.01 g The saturation coupling amount of magnetic chitosan microspheres to the antibody is 25 ~ 50 μL. When the E. sakazakii concentration was low, the prepared immunomagnetic chitosan microspheres had a capture rate of 94.7% against Enterobacter sakazakii and a sensitivity of 5 cfu / m L to E. sakazakii. Conclusion: Enterobacter sakazakii immune magnetic chitosan microspheres are obtained, and the microspheres are an effective method for rapid enrichment and isolation of E. sakazakii.