基于金纳米颗粒的呼吸道合胞病毒及其细胞膜受体免疫分析新方法研究
发布时间：2018-09-05 10:51
【摘要】：呼吸道合胞病毒(respiratory syncytial virus, RSV)为副粘病毒科肺炎病毒属,有包膜的非节段性单股负链RNA病毒,是引起下呼吸道感染最重要的病原体之一。RSV感染易导致毛细支气管炎、肺炎、慢性阻塞性肺炎等疾病,主要影响婴幼儿、老人及免疫缺陷的成人,具有极高的发病率和死亡率。到目前为止,尚无安全有效的疫苗可预防RSV感染。因此,建立简单、快速、灵敏的RSV检测方法对于疾病的早期诊断、治疗及预防控制均具有非常重要的研究意义。此外,研究RSV与宿主细胞之间的相互作用,对于寻求新的药物靶点,开发RSV治疗药物也具有十分重要的研究意义。本文将金纳米材料引入传统的免疫分析,结合信号放大技术,建立了新型的RSV及其细胞膜受体免疫分析方法。具体的研究内容如下：1、基于金纳米颗粒的RSV免疫分析1)以金纳米颗粒(AuNPs)作为载体,建立了一种信号放大的RSV免疫新方法。柠檬酸包被的负电AuNPs通过静电吸附与抗体和碱性磷酸酶(ALP)结合,制备了双标记AuNPs复合物。另外,通过生物素-亲和素的识别作用,将抗体修饰到磁微米表面,制备了免疫磁珠。在检测过程中,免疫磁珠、病毒和双标记AuNPs形成三明治结构,通过ALP催化底物显色实现了对病毒的分析,检测范围为0.5-80pg/mL。与传统的免疫分析方法相比,AuNPs可负载多个酶分子,增强了检测信号,提高了检测灵敏度,有望进一步用于其他病原体的临床诊断及治疗情况的监测。2)以AuNPs作为信号报告分子,结合双重信号放大技术构建了一种高灵敏的RSV等离子免疫分析方法。三磷酸腺苷(ATP)带有负电荷,可使正电AuNPs聚集；而在ALP作用下,ATP可脱磷酸化为不带电荷的腺苷分子,不诱导AuNPs聚集,即ALP可促使AuNPs分散。基于此设计了一种新型的等离子免疫方法,同时,引入两种信号放大方式,利用磁微米实现酶的多标记并利用金属离子增强酶催化反应,显著增强了检测信号。该方法的线性范围为0.1-30 pg/mL,检测限达O.02 pg/mL,与传统方法相比,灵敏度得到进一步提高,具有潜在应用价值。2、金纳米颗粒放大的RSV宿主细胞膜受体原位免疫分析研究利用金纳米放大技术,对宿主细胞表面Toll样受体(TLR)进行原位免疫分析。RSV感染宿主细胞时,TLR可识别并结合相关配体,激活下游信号传导,诱发一系列级联反应,诱导炎症反应并刺激TLR受体表达。以宿主细胞作为传感元件,利用抗原-抗体特异性识别反应分析TLR含量的变化,并研究其与RSV活力之间的关系。结果表明HEp-2细胞的TLR4表达量与RSV的感染复数(MOI)值之间不存在明显的线性关系。由于细胞对外界刺激具有高度敏感性,细胞膜受体原位免疫分析研究可观察RSV与宿主细胞间的相互作用。综上,本论文成功将金纳米材料与传统的免疫分析结合,建立了RSV及其细胞膜受体免疫分析新方法。采用信号放大技术实现了检测灵敏度的提高,扩展了金纳米材料在免疫分析中的应用,同时将细胞作为传感单元对膜受体进行原位免疫分析,为病原微生物与宿主之间相互作用的研究提供了新的思路。
[Abstract]:Respiratory syncytial virus (RSV), a non-segmental single stranded negative strand RNA virus with envelope, is one of the most important pathogens causing lower respiratory tract infection. Up to now, there is no safe and effective vaccine to prevent RSV infection. Therefore, it is very important to establish a simple, rapid and sensitive RSV detection method for early diagnosis, treatment, prevention and control of diseases. In this paper, the gold nanoparticles were introduced into the traditional immunoassay, and a new immunoassay method for RSV and its cell membrane receptors was established by combining the signal amplification technique. Epidemic analysis 1) A novel RSV immunoassay method using gold nanoparticles (AuNPs) as carriers was developed. The negative AuNPs coated with citric acid were combined with antibodies and alkaline phosphatase (ALP) by electrostatic adsorption to prepare double-labeled AuNPs complexes. Immunomagnetic beads were prepared. In the process of detection, immunomagnetic beads, viruses and double-labeled AuNPs formed sandwich structure. The detection range of the virus was 0.5-80pg/mL. Compared with the traditional immunoassay method, AuNPs could load many enzyme molecules, enhance the detection signal and improve the detection sensitivity. It is expected to be further used in clinical diagnosis and treatment monitoring of other pathogens. 2) A highly sensitive RSV plasma immunoassay method was developed by using AuNPs as signal reporter molecule and double signal amplification technique. A novel plasma immune method based on ALP was designed. Two signal amplification modes were introduced. Magnetic micron was used to realize enzyme multi-labeling and metal ion was used to enhance enzyme catalytic reaction. In situ immunoassay of host cell membrane receptors amplified by gold nanoparticles using gold nanoparticles in situ immunoassay for Toll-like receptors (TLR) on the surface of host cells using gold nanoparticle amplification technique. In the host cell, TLR can recognize and bind to related ligands, activate downstream signal transduction, induce a series of cascade reactions, induce inflammation and stimulate the expression of TLR receptor. The changes of TLR content in the host cell were analyzed by antigen-antibody specific recognition reaction, and the relationship between TLR content and RSV activity was studied. There is no obvious linear relationship between the expression of TLR4 and the infection complex (MOI) of RSV. Because cells are highly sensitive to external stimuli, in situ immunoassay of cell membrane receptors can observe the interaction between RSV and host cells. A new method for immunoassay of RSV and its membrane receptors was developed. The sensitivity of detection was improved by signal amplification technique. The application of gold nanomaterials in immunoassay was expanded. At the same time, cells were used as sensing units for in situ immunoassay of membrane receptors. Thinking.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6
[Abstract]:Respiratory syncytial virus (RSV), a non-segmental single stranded negative strand RNA virus with envelope, is one of the most important pathogens causing lower respiratory tract infection. Up to now, there is no safe and effective vaccine to prevent RSV infection. Therefore, it is very important to establish a simple, rapid and sensitive RSV detection method for early diagnosis, treatment, prevention and control of diseases. In this paper, the gold nanoparticles were introduced into the traditional immunoassay, and a new immunoassay method for RSV and its cell membrane receptors was established by combining the signal amplification technique. Epidemic analysis 1) A novel RSV immunoassay method using gold nanoparticles (AuNPs) as carriers was developed. The negative AuNPs coated with citric acid were combined with antibodies and alkaline phosphatase (ALP) by electrostatic adsorption to prepare double-labeled AuNPs complexes. Immunomagnetic beads were prepared. In the process of detection, immunomagnetic beads, viruses and double-labeled AuNPs formed sandwich structure. The detection range of the virus was 0.5-80pg/mL. Compared with the traditional immunoassay method, AuNPs could load many enzyme molecules, enhance the detection signal and improve the detection sensitivity. It is expected to be further used in clinical diagnosis and treatment monitoring of other pathogens. 2) A highly sensitive RSV plasma immunoassay method was developed by using AuNPs as signal reporter molecule and double signal amplification technique. A novel plasma immune method based on ALP was designed. Two signal amplification modes were introduced. Magnetic micron was used to realize enzyme multi-labeling and metal ion was used to enhance enzyme catalytic reaction. In situ immunoassay of host cell membrane receptors amplified by gold nanoparticles using gold nanoparticles in situ immunoassay for Toll-like receptors (TLR) on the surface of host cells using gold nanoparticle amplification technique. In the host cell, TLR can recognize and bind to related ligands, activate downstream signal transduction, induce a series of cascade reactions, induce inflammation and stimulate the expression of TLR receptor. The changes of TLR content in the host cell were analyzed by antigen-antibody specific recognition reaction, and the relationship between TLR content and RSV activity was studied. There is no obvious linear relationship between the expression of TLR4 and the infection complex (MOI) of RSV. Because cells are highly sensitive to external stimuli, in situ immunoassay of cell membrane receptors can observe the interaction between RSV and host cells. A new method for immunoassay of RSV and its membrane receptors was developed. The sensitivity of detection was improved by signal amplification technique. The application of gold nanomaterials in immunoassay was expanded. At the same time, cells were used as sensing units for in situ immunoassay of membrane receptors. Thinking.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6
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