【作者】 王颖；

【导师】 王克霞；

【作者基本信息】 安徽理工大学 ， 医学免疫学， 2011， 硕士

【摘要】 肠道病毒71型(EV71)属于小核糖核酸病毒,感染后可引起手足口病(hand-foot-mouth disease,HFMD),部分患儿可引起一系列中枢神经系统并发症,出现重症病例和死亡病例。因此,建立一种可以快速均相检测EV71 IgM抗体的方法在HFMD早期诊断中显得尤为重要。根据HFMD发病特点,我们建立了具有高敏感性、精确性、均一性、背景低、广泛的动态检测范围、无需洗涤及样本需求量极少等特点的针对EV71 IgM的AlphaLISA检测技术。在本实验中,我们成功的表达纯化了EV71 VP1 1-297及EV71 VP1 1-177重组蛋白,经鉴定后将抗原生物素化。通过ELISA法摸索出生物素化的EV71 VP1的最适量为37.5 ng。优化供体微珠、受体微珠、血清量等实验条件,初步建立了基于VP1的EV71 IgM抗体的AlphaLISA检测方法。我们首先使用ELISA法对10份健康人的血清和30份手足口病病人(30份手足口病病人核酸检测EV71均为阳性)的血清进行评价,健康人的血清中EV71 IgM检测均为阴性,30份手足口病病人的血清中EV71 IgM阳性为16份。然后用建立的AlphaLISA方法体系对上述样本进行检测,检测结果显示：在10个健康人中,AlphaLISA方法检测EV71 IgM也为阴性。在30份手足口病病人血清中,ELISA方法检测出的16份EV71 IgM阳性的样本,AlphaLISA方法检测EV71 IgM均为阳性,然而AlphaLISA方法在ELISA法检测为阴性的血清中检出6个IgM阳性,符合率达到100%,初步建立了均相、快速检测EV71 IgM的平台。更多还原

【Abstract】 Enterovirus 71 (EV71), a member of the family Picornaviridae, is known as one of the major causative agents of hand, foot and mouth disease(HFMD), and sometimes associated with a series of central nervous system diseases in some infected children so much as lead to severe cases and death. Accordingly, developing a quick homogeneous alternative approach to human IgM detection looks so crucial for early diagnosis of HFMD.According to the characteristic of HFMD, we established a method for EV71 IgM detection which is called AlphaLISA technology with high sensitivity, specificity, homogeneous, low background, wide dynamic ranges, no-wash steps and low sample volumes.In this study, we successfully purified and verified the recombinant protein EV71 VP1 1-297 and protein EV71 VP1 1-177. After proteins were biotinylated, the optimal quantities of the antigen was determinated by ELISA, and the lowest protein quantity finally is 37.5ng per well. Adjusting acceptor beads, dornor beads, Bio-protein and volume of serum, AlphaLISA reaction system was optimized in total assay volume 20 ul containing serum 2μl.Subsequently, we detected the serum samples in 10 healthy subjects with commercial ELISA IgM kits and 30 samples from the patients of HFMD with AlphaLISA, respectively, for verifying the protocol newly established. The results revealed IgM negative for the total 10 healthy subjects by ELISA as well as AlphaLISA. of the 30 HFMD patients, positive serum IgM was detected in 16 with respectively ELISA and AlphaLISA, and yet, additional detection of the negative samples among the 10 healthy subjects by ELISA revealed another 6 IgM positive with AlphaLISA. The results of the AlphaLISA assay are coincident with ELISA kits, suggesting that AlphaLISA can be competent for EV71 IgM serum detection in patients of HFMD.更多还原