肠道病毒68型（EV-D68）抗原检测ELISA方法的建立
发布时间：2018-11-12 18:16
【摘要】：[背景]肠道病毒68型(Enterovirus D68,EV-D68)于1962年首次分离自患有肺炎和支气管炎的住院儿童。EV-D68属于病毒科,肠道病毒属,肠道病毒D组。自被发现后关于EV-D68的报道甚少,直至2005年后才开始大规模的爆发。北美于2014年爆发了有史以来最大规模的一次EV-D68流行。EV-D68感染儿童可以引起咳嗽、喘息和血氧不足等急性呼吸道疾病。最近爆发的急性驰缓性麻痹也被怀疑与EV-D68感染相关。因此,尽早的建立一种方便快速检测EV-D68的ELISA方法十分有必要。[方法]本课题研究利用细胞工厂大规模的培养Vero细胞后进行EV-D68病毒的扩增,从而得到较多的病毒原液。利用分子量为8000的聚乙二醇(PEG8000)进行浓缩,抽提后,采用碘克沙醇进行密度梯度离心,得到纯化的抗原,之后进行免疫兔子。隔段时间采血,检测中和抗体水平,在中和抗体处于较高水平时,对兔子进行颈动脉取血。利用亲和层析法进行血清IgG的纯化,产物经过SDS-PAGE鉴定后,分取一部分用生物素(Biotin)进行标记作为检测抗体,剩下的没有生物素化的抗体作为捕获抗体。[结果]运用矩阵法,利用上述的捕获抗体与检测抗体,检测抗原为EV-D68病毒,阴性对照选择EV71病毒液与Vero细胞冻存液。不断优化包被抗体量、孵育时间、检测抗体浓度以及亲和素酶浓度。最终初步建立一套ELISA体系用来检测肠道病毒68型。[结论]根据《中国药典》相关规定,测定上述ELISA方法的变异系数及其稳定性,并且进行灵敏度的测定和特异性的检测,最终初步建立了一种检测EV-D68病毒的ELISA方法,为以后的EV-D68病毒标准化检测试剂盒提供了强有力的基础。
[Abstract]:[background] Enterovirus 68 (Enterovirus D68 EV-D68) was first isolated from hospitalized children with pneumonia and bronchitis in 1962. EV-D68 belongs to viridae, enterovirus genus, enterovirus group D. There have been very few reports of EV-D68 since its discovery, and a major outbreak did not begin until 2005. North America witnessed the largest EV-D68 epidemic ever recorded in 2014. Children infected with EV-D68 can cause acute respiratory diseases such as cough, wheezing and lack of blood oxygen. The recent outbreak of acute flaccid paralysis is also suspected to be associated with EV-D68 infection. Therefore, it is necessary to establish a ELISA method for rapid detection of EV-D68 as soon as possible. [methods] A large scale culture of Vero cells in a cell factory was carried out to amplify the EV-D68 virus. Polyethylene glycol (PEG8000) with molecular weight of 8000 was used to concentrate. After extraction, the purified antigen was obtained by density gradient centrifugation with iodoxacin, and then the rabbits were immunized. Blood samples were collected at intervals to detect neutralization antibody level and carotid artery blood was taken from rabbits when neutralizing antibody was at a higher level. The serum IgG was purified by affinity chromatography. After being identified by SDS-PAGE, some of the products were labeled with biotinylated (Biotin) as detection antibodies, and the remaining antibodies without biotin were used as capture antibodies. [results] using the matrix method, the EV-D68 virus antigen was detected by using the captured antibody and the detection antibody mentioned above. The negative control group chose the EV71 virus solution and the Vero cell cryopreservation solution. Continue to optimize the amount of coated antibody, incubation time, detection of antibody concentration and affinity enzyme concentration. Finally, a set of ELISA system was established to detect enterovirus 68. [conclusion] according to the relevant provisions of the Chinese Pharmacopoeia, the variation coefficient and stability of the ELISA method mentioned above were determined, and the sensitivity and specificity were determined. Finally, a ELISA method for the detection of EV-D68 virus was preliminarily established. It provides a strong basis for the standardization of EV-D68 virus detection kit.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.2
[Abstract]:[background] Enterovirus 68 (Enterovirus D68 EV-D68) was first isolated from hospitalized children with pneumonia and bronchitis in 1962. EV-D68 belongs to viridae, enterovirus genus, enterovirus group D. There have been very few reports of EV-D68 since its discovery, and a major outbreak did not begin until 2005. North America witnessed the largest EV-D68 epidemic ever recorded in 2014. Children infected with EV-D68 can cause acute respiratory diseases such as cough, wheezing and lack of blood oxygen. The recent outbreak of acute flaccid paralysis is also suspected to be associated with EV-D68 infection. Therefore, it is necessary to establish a ELISA method for rapid detection of EV-D68 as soon as possible. [methods] A large scale culture of Vero cells in a cell factory was carried out to amplify the EV-D68 virus. Polyethylene glycol (PEG8000) with molecular weight of 8000 was used to concentrate. After extraction, the purified antigen was obtained by density gradient centrifugation with iodoxacin, and then the rabbits were immunized. Blood samples were collected at intervals to detect neutralization antibody level and carotid artery blood was taken from rabbits when neutralizing antibody was at a higher level. The serum IgG was purified by affinity chromatography. After being identified by SDS-PAGE, some of the products were labeled with biotinylated (Biotin) as detection antibodies, and the remaining antibodies without biotin were used as capture antibodies. [results] using the matrix method, the EV-D68 virus antigen was detected by using the captured antibody and the detection antibody mentioned above. The negative control group chose the EV71 virus solution and the Vero cell cryopreservation solution. Continue to optimize the amount of coated antibody, incubation time, detection of antibody concentration and affinity enzyme concentration. Finally, a set of ELISA system was established to detect enterovirus 68. [conclusion] according to the relevant provisions of the Chinese Pharmacopoeia, the variation coefficient and stability of the ELISA method mentioned above were determined, and the sensitivity and specificity were determined. Finally, a ELISA method for the detection of EV-D68 virus was preliminarily established. It provides a strong basis for the standardization of EV-D68 virus detection kit.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.2
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