基于金納米顆粒的呼吸道合胞病毒及其細(xì)胞膜受體免疫分析新方法研究
發(fā)布時(shí)間：2018-09-05 10:51
【摘要】：呼吸道合胞病毒(respiratory syncytial virus, RSV)為副粘病毒科肺炎病毒屬,有包膜的非節(jié)段性單股負(fù)鏈RNA病毒,是引起下呼吸道感染最重要的病原體之一。RSV感染易導(dǎo)致毛細(xì)支氣管炎、肺炎、慢性阻塞性肺炎等疾病,主要影響嬰幼兒、老人及免疫缺陷的成人,具有極高的發(fā)病率和死亡率。到目前為止,尚無(wú)安全有效的疫苗可預(yù)防RSV感染。因此,建立簡(jiǎn)單、快速、靈敏的RSV檢測(cè)方法對(duì)于疾病的早期診斷、治療及預(yù)防控制均具有非常重要的研究意義。此外,研究RSV與宿主細(xì)胞之間的相互作用,對(duì)于尋求新的藥物靶點(diǎn),開發(fā)RSV治療藥物也具有十分重要的研究意義。本文將金納米材料引入傳統(tǒng)的免疫分析,結(jié)合信號(hào)放大技術(shù),建立了新型的RSV及其細(xì)胞膜受體免疫分析方法。具體的研究?jī)?nèi)容如下：1、基于金納米顆粒的RSV免疫分析1)以金納米顆粒(AuNPs)作為載體,建立了一種信號(hào)放大的RSV免疫新方法。檸檬酸包被的負(fù)電AuNPs通過(guò)靜電吸附與抗體和堿性磷酸酶(ALP)結(jié)合,制備了雙標(biāo)記AuNPs復(fù)合物。另外,通過(guò)生物素-親和素的識(shí)別作用,將抗體修飾到磁微米表面,制備了免疫磁珠。在檢測(cè)過(guò)程中,免疫磁珠、病毒和雙標(biāo)記AuNPs形成三明治結(jié)構(gòu),通過(guò)ALP催化底物顯色實(shí)現(xiàn)了對(duì)病毒的分析,檢測(cè)范圍為0.5-80pg/mL。與傳統(tǒng)的免疫分析方法相比,AuNPs可負(fù)載多個(gè)酶分子,增強(qiáng)了檢測(cè)信號(hào),提高了檢測(cè)靈敏度,有望進(jìn)一步用于其他病原體的臨床診斷及治療情況的監(jiān)測(cè)。2)以AuNPs作為信號(hào)報(bào)告分子,結(jié)合雙重信號(hào)放大技術(shù)構(gòu)建了一種高靈敏的RSV等離子免疫分析方法。三磷酸腺苷(ATP)帶有負(fù)電荷,可使正電AuNPs聚集；而在ALP作用下,ATP可脫磷酸化為不帶電荷的腺苷分子,不誘導(dǎo)AuNPs聚集,即ALP可促使AuNPs分散�；诖嗽O(shè)計(jì)了一種新型的等離子免疫方法,同時(shí),引入兩種信號(hào)放大方式,利用磁微米實(shí)現(xiàn)酶的多標(biāo)記并利用金屬離子增強(qiáng)酶催化反應(yīng),顯著增強(qiáng)了檢測(cè)信號(hào)。該方法的線性范圍為0.1-30 pg/mL,檢測(cè)限達(dá)O.02 pg/mL,與傳統(tǒng)方法相比,靈敏度得到進(jìn)一步提高,具有潛在應(yīng)用價(jià)值。2、金納米顆粒放大的RSV宿主細(xì)胞膜受體原位免疫分析研究利用金納米放大技術(shù),對(duì)宿主細(xì)胞表面Toll樣受體(TLR)進(jìn)行原位免疫分析。RSV感染宿主細(xì)胞時(shí),TLR可識(shí)別并結(jié)合相關(guān)配體,激活下游信號(hào)傳導(dǎo),誘發(fā)一系列級(jí)聯(lián)反應(yīng),誘導(dǎo)炎癥反應(yīng)并刺激TLR受體表達(dá)。以宿主細(xì)胞作為傳感元件,利用抗原-抗體特異性識(shí)別反應(yīng)分析TLR含量的變化,并研究其與RSV活力之間的關(guān)系。結(jié)果表明HEp-2細(xì)胞的TLR4表達(dá)量與RSV的感染復(fù)數(shù)(MOI)值之間不存在明顯的線性關(guān)系。由于細(xì)胞對(duì)外界刺激具有高度敏感性,細(xì)胞膜受體原位免疫分析研究可觀察RSV與宿主細(xì)胞間的相互作用。綜上,本論文成功將金納米材料與傳統(tǒng)的免疫分析結(jié)合,建立了RSV及其細(xì)胞膜受體免疫分析新方法。采用信號(hào)放大技術(shù)實(shí)現(xiàn)了檢測(cè)靈敏度的提高,擴(kuò)展了金納米材料在免疫分析中的應(yīng)用,同時(shí)將細(xì)胞作為傳感單元對(duì)膜受體進(jìn)行原位免疫分析,為病原微生物與宿主之間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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.6
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