【作者】 陈伶利；

【导师】 邓乐；

【作者基本信息】 湖南师范大学 ， 微生物学， 2005， 硕士

【摘要】 本文建立了一种金黄色葡萄球菌及其SPA快速分离检验的新方法—IMS-MS-SPR,通过这种方法,能对样品中的金黄色葡萄球菌及SPA快速分离后定性定量检测。 通过乳液聚合法,合成一系列不同粒径的Fe₃O₄磁核,再经苯乙烯—丙烯醛共聚物为高分子壳层,合成了带醛基的高分子磁珠。所得磁珠外观成很规则的球形,表面光滑,粒径较为均一,介于0.6μ～1μ之间;并用红外光谱仪和电导滴定法对磁珠表面醛基进行了定性和定量测定,经电导率仪测定醛基含量达到0.7547mmol/g。 将人IgG定向结合到制备的磁珠上,得到免疫磁珠。取适量该免疫磁珠对金黄色葡萄球菌进行分离;并对磁珠分离混合菌液中的菌的纯培养物进行生理生化鉴定。 用制备的免疫磁珠从金黄色葡萄球菌细胞粉碎液中分离SPA蛋白,洗脱之后所得样品用MALDI-TOF/MS进行质谱分析。采用BIA进行人IgG与金黄色葡萄球菌相互作用的实时检测。 本方法及技术可以在2h内分离出样品中的致病性金黄色葡萄球菌或者其细胞中的SPA,具有高度的特异性,操作简单,为致病性金黄色葡萄球菌的快速分离开辟了一条新的道路。同时通过对BIA/MS联用技术的采用,能在短时间内对分离出来的致病性金黄色葡萄球菌进行鉴定以及对其浓度的检测,从而建立了一系列完整的致病性金黄色葡萄球菌快速分离鉴定以及浓度测定的方法。如果在磁珠表面接上不同的抗体,能分离其它的细胞和蛋白。此方法有望在临床检验以及微生物检测等方面得到大力应用。更多还原

【Abstract】 The aim of this study was to establish a IMS-MS-SPR method for the detection of S. aureus and its SpA at the same time, which is consisted of isolating S. aureus and trapping-enrichmenting its SpA by IMS, and then the end point is determined using either MS or SPR measurements. Magnetic beads(MB) containing aldehyde group were synthesized with latex-polymerization, they are regular in shape and identical in diameter, with content of the aldehyde group of 0.7547 mmol/g, and its surface are modified with oriented human IgG in covalent linkage. Immune magnetic beads(1MB) should be obtained. As soon as sample of pulverat-treated bacterial cell lysate (108cfu/ml) were incubated with IMB at 4℃ for 30min, the protein A were captured and separated from the mixed solution in a few minutes by the IMB in the magnetic field and then detected using mass spectrometry after washed. SPR was also used to detect the interaction of IgG and SpA on the cells in situ. All in all, this mehod could be employed to detect rapidly SpA and S. aureus within 2h and also be applied to detect other cells or their membrane proteins with changed modified antibodies.更多还原