腸道病毒68型（EV-D68）抗原檢測(cè)ELISA方法的建立
發(fā)布時(shí)間：2018-11-12 18:16
【摘要】：[背景]腸道病毒68型(Enterovirus D68,EV-D68)于1962年首次分離自患有肺炎和支氣管炎的住院兒童。EV-D68屬于病毒科,腸道病毒屬,腸道病毒D組。自被發(fā)現(xiàn)后關(guān)于EV-D68的報(bào)道甚少,直至2005年后才開(kāi)始大規(guī)模的爆發(fā)。北美于2014年爆發(fā)了有史以來(lái)最大規(guī)模的一次EV-D68流行。EV-D68感染兒童可以引起咳嗽、喘息和血氧不足等急性呼吸道疾病。最近爆發(fā)的急性馳緩性麻痹也被懷疑與EV-D68感染相關(guān)。因此,盡早的建立一種方便快速檢測(cè)EV-D68的ELISA方法十分有必要。[方法]本課題研究利用細(xì)胞工廠大規(guī)模的培養(yǎng)Vero細(xì)胞后進(jìn)行EV-D68病毒的擴(kuò)增,從而得到較多的病毒原液。利用分子量為8000的聚乙二醇(PEG8000)進(jìn)行濃縮,抽提后,采用碘克沙醇進(jìn)行密度梯度離心,得到純化的抗原,之后進(jìn)行免疫兔子。隔段時(shí)間采血,檢測(cè)中和抗體水平,在中和抗體處于較高水平時(shí),對(duì)兔子進(jìn)行頸動(dòng)脈取血。利用親和層析法進(jìn)行血清IgG的純化,產(chǎn)物經(jīng)過(guò)SDS-PAGE鑒定后,分取一部分用生物素(Biotin)進(jìn)行標(biāo)記作為檢測(cè)抗體,剩下的沒(méi)有生物素化的抗體作為捕獲抗體。[結(jié)果]運(yùn)用矩陣法,利用上述的捕獲抗體與檢測(cè)抗體,檢測(cè)抗原為EV-D68病毒,陰性對(duì)照選擇EV71病毒液與Vero細(xì)胞凍存液。不斷優(yōu)化包被抗體量、孵育時(shí)間、檢測(cè)抗體濃度以及親和素酶濃度。最終初步建立一套ELISA體系用來(lái)檢測(cè)腸道病毒68型。[結(jié)論]根據(jù)《中國(guó)藥典》相關(guān)規(guī)定,測(cè)定上述ELISA方法的變異系數(shù)及其穩(wěn)定性,并且進(jìn)行靈敏度的測(cè)定和特異性的檢測(cè),最終初步建立了一種檢測(cè)EV-D68病毒的ELISA方法,為以后的EV-D68病毒標(biāo)準(zhǔn)化檢測(cè)試劑盒提供了強(qiáng)有力的基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373.2
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