本文關鍵詞： 結(jié)核分枝桿菌 γ-干擾素釋放試驗 酶聯(lián)免疫斑點技術 酶聯(lián)免疫法 化學發(fā)光法　出處：《河南工業(yè)大學》2014年碩士論文　論文類型：學位論文

【摘要】：結(jié)核病(tuberculosis,TB)是由結(jié)核分枝桿菌(mycobacterium tuberculosis,MTB)引起的傳染性疾病，結(jié)核病的臨床診斷方法主要分為三大類:細菌學檢查、分子生物學檢查和免疫學檢查，其中免疫學檢查以準確性高、操作簡便而備受關注。近年來，免疫學檢查中γ-干擾素（IFN-γ）釋放試驗(Interferon Gamma ReleaseAssay, IGRA)因高靈敏度和特異性而引起了人們的興趣。IGRA基于酶聯(lián)免疫斑點試驗（ELISPOT）最早被廣泛應用，世界衛(wèi)生組織（WHO）推薦兩種結(jié)核病診斷試劑QFT-G試劑盒和T-SPOT.TB試劑盒均基于ELISPOT。 IGRA是采用酶聯(lián)免疫吸附實驗（ELISA）方法定量檢測全血在結(jié)核分枝桿菌特異性抗原刺激下釋放IFN-γ的水平，，用于診斷結(jié)核分枝桿菌感染以及結(jié)核病。其檢測原理是采用夾心法原理進行檢測，衡量特異性抗原介導的細胞免疫反應強度。通過結(jié)核分枝桿菌特異性抗原刺激結(jié)核分枝桿菌感染者特異性T淋巴細胞并使其增殖，釋放IFN-γ。用IFN-γ抗體包被磁微粒，辣根過氧化物酶標記IFN-γ抗體制備酶結(jié)合物。通過免疫反應形成抗體-抗原-抗體復合物，該復合物催化發(fā)光底物發(fā)出光子，發(fā)光強度與IFN-γ的含量成正比。 本研究基于IGRA建立一種化學發(fā)光方法，并將其應用于結(jié)核病診斷�；瘜W發(fā)光方法通過磁微粒混懸液制備，酶結(jié)合物制備，校準品制備，反應體系優(yōu)化，正常參考值確定，穩(wěn)定性研究建立而成。該方法應用于結(jié)核病診斷的研究主要通過與臨床確診信息對比，考核化學發(fā)光方法應用于結(jié)核病診斷的靈敏度、特異性和準確性指標，以OxfordImmunotec Ltd生產(chǎn)的T-SPOT.TB試劑盒作對照，考核化學發(fā)光方法應用于結(jié)核病診斷與T-SPOT.TB試劑盒的陽性符合率、陰性符合率和總符合率。建立的化學發(fā)光方法將檢測時間縮短為30分鐘，該化學發(fā)光方法應用于結(jié)核病診斷中，與臨床確診結(jié)果對比，靈敏度為86.19%（69/81），特異性為84.26%（589/699），準確性為84.36%（658/780）。以T-SPOT.TB試劑盒作標桿，該化學發(fā)光方法應用于結(jié)核病診斷與T-SPOT.TB試劑盒的陽性符合率為85.88%（225/262），陰性符合率為81.44%（215/264），總符合率為83.65%（440/526）。 本研究通過建立化學發(fā)光方法應用于結(jié)核病的診斷，縮短了檢測時間，提高了檢測線性范圍，更加靈敏和特異，并且對環(huán)境沒有污染。為今后結(jié)核病診斷使用化學發(fā)光方法奠定基礎。
[Abstract]:Tuberculosis tuberculosis (TB) is a kind of infectious disease caused by Mycobacterium tuberculosisus MTB (Mycobacterium tuberculosis). The methods of clinical diagnosis of tuberculosis are divided into three main categories: bacteriological examination, molecular biological examination and immunological examination, among which immunological examination has attracted much attention due to its high accuracy and simple operation. Interferon Gamma ReleaseAssay (IFN- 緯) release test in immunological examination. IGRA-based enzyme-linked immunoblot assay (ElisPOT) has been widely used in the first place because of its high sensitivity and specificity. The World Health Organization (WHO) recommended two TB diagnostic kits, QFT-G Kit and T-SPOT.TB Kit, both based on ElisPOT. Enzyme linked immunosorbent assay (Elisa) was used to detect the level of IFN- 緯 in whole blood stimulated by specific antigen of Mycobacterium tuberculosis. Used in the diagnosis of Mycobacterium tuberculosis infection and tuberculosis. To measure the intensity of cellular immune response mediated by specific antigen. The specific T lymphocytes of Mycobacterium tuberculosis infected patients were stimulated and proliferated by the specific antigen of Mycobacterium tuberculosis. IFN- 緯 was released. The magnetic particles were coated with IFN- 緯 antibody, and the IFN- 緯 antibody was labeled with horseradish peroxidase to prepare the enzyme conjugate. The antibody antigen-antibody complex was formed by immune reaction. The luminescence intensity is proportional to the content of IFN- 緯. In this study, a chemiluminescence method based on IGRA was established and applied to the diagnosis of tuberculosis. The chemiluminescence method was prepared by magnetic particle suspension, enzyme conjugate preparation, calibration preparation, and reaction system optimization. The normal reference value was determined and the stability study was established. The sensitivity of the chemiluminescence method applied to the diagnosis of tuberculosis was evaluated by comparing with the clinical diagnosis information. The specificity and accuracy were compared with T-SPOT.TB kit produced by OxfordImmunotec Ltd. The positive coincidence rate, negative coincidence rate and total coincidence rate of the chemiluminescence method applied to the diagnosis of tuberculosis and T-SPOT.TB kit were evaluated. The established chemiluminescence method shortened the detection time to 30 minutes. The chemiluminescence method was applied to the diagnosis of tuberculosis. Compared with the results of clinical diagnosis, the sensitivity of the chemiluminescence method was 86.19 / 89 / 89 and the specificity was 84.26 / 589 / 699). The accuracy was 84.36 / 7800.The T-SPOT.TB kit was used as a benchmark. The positive coincidence rate of the chemiluminescence method applied to the diagnosis of tuberculosis and T-SPOT.TB kit was 85.88 / 225 / 262). The negative coincidence rate was 81.44 / 264% and the total coincidence rate was 83.65% and 440% / 526% respectively. In this study, a chemiluminescence method was established for the diagnosis of tuberculosis. The detection time was shortened, the linear range of detection was improved, and the detection was more sensitive and specific. And no pollution to the environment. It lays the foundation for the use of chemiluminescence in the diagnosis of tuberculosis in the future.
【學位授予單位】：河南工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R52

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