鼠伤寒沙门菌免疫磁珠的构建及应用效果评价

Evaluation of the construction and application effect of Salmonella typhimurium immunomagnetic beads

【目的】制备鼠伤寒沙门菌高免血清并纯化抗体,构建免疫磁珠,进行吸附效果评价,为建立鼠伤寒沙门菌快速检测方法奠定基础。【方法】利用试验兔制备高免血清,检测效价后纯化提取IgG抗体,纯化后的抗体采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺方法与四氧化三铁磁性纳米颗粒偶联,制备出鼠伤寒沙门菌免疫磁珠。利用抗原抗体反应原理,通过免疫磁珠对样品中的鼠伤寒沙门菌进行富集分离,将鼠伤寒沙门菌捕获到磁珠表面,达到检测鼠伤寒沙门菌的目的。【结果】制备出的血清效价达1∶51 200,纯化后的抗体特异性强,仅与鼠伤寒沙门菌有凝集反应,与大肠杆菌、金黄色葡萄球菌、单增李斯特菌均无凝集反应;制备的免疫磁珠能够富集奶样样品、肉样样品与蛋黄样品中的鼠伤寒沙门菌,灵敏度为101CFU/mL。【结论】该研究制备的鼠伤寒沙门菌免疫磁珠,能够有效富集各种样品基质中的鼠伤寒沙门菌,有望用于鼠伤寒沙门菌快速检测方法的建立。

[Objective] To prepare the high immune serum of Salmonella typhimurium and purify the antibody, and construct the immune magnetic beads, laying the foundation for the further establishment of rapid detection method of Salmonella typhimurium.[Methods] Hyperimmune serum was prepared from experimental rabbits. After detection of titer, IgG antibodies were purified and extracted. The purified antibody was conjugated with 1-(3-dimethylaminopropyl)-3-ethylcarbondiimide hydrochloride and N-hydroxysuccinimide nanoparticles to prepare Salmonella typhimurium immune magnetic beads. Using the principle of antigen-antibody reaction, the Salmonella typhimurium in the sample was enriched and separated by immunomagnetic beads, and the Salmonella typhimurium was captured on the surface of magnetic beads, so as to achieve the purpose of Salmonella typhimurium detection. [Results] The serum titer was 1∶51 200. The purified antibodies agglutinate only with Salmonella typhimurium. There was no agglutination reaction with Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The prepared immunomagnetic beads could enrich Salmonella typhimurium in milk samples, meat samples and egg yolks with sensitivity of 10~1 CFU/mL. [Conclusion] The Salmonella typhimurium immunomagnetic beads prepared in this study can effectively enrich Salmonella typhimurium in various sample substrates, which is expected to be used for the establishment of a rapid detection method for Salmonella typhimurium.