化学发光方法的建立及其应用于结核病诊断的研究

发布时间：2018-01-27 04:01

本文关键词： 结核分枝杆菌 γ-干扰素释放试验酶联免疫斑点技术酶联免疫法化学发光法　出处：《河南工业大学》2014年硕士论文　论文类型：学位论文

【摘要】：结核病(tuberculosis,TB)是由结核分枝杆菌(mycobacterium tuberculosis,MTB)引起的传染性疾病，结核病的临床诊断方法主要分为三大类:细菌学检查、分子生物学检查和免疫学检查，其中免疫学检查以准确性高、操作简便而备受关注。近年来，免疫学检查中γ-干扰素（IFN-γ）释放试验(Interferon Gamma ReleaseAssay, IGRA)因高灵敏度和特异性而引起了人们的兴趣。IGRA基于酶联免疫斑点试验（ELISPOT）最早被广泛应用，世界卫生组织（WHO）推荐两种结核病诊断试剂QFT-G试剂盒和T-SPOT.TB试剂盒均基于ELISPOT。 IGRA是采用酶联免疫吸附实验（ELISA）方法定量检测全血在结核分枝杆菌特异性抗原刺激下释放IFN-γ的水平，，用于诊断结核分枝杆菌感染以及结核病。其检测原理是采用夹心法原理进行检测，衡量特异性抗原介导的细胞免疫反应强度。通过结核分枝杆菌特异性抗原刺激结核分枝杆菌感染者特异性T淋巴细胞并使其增殖，释放IFN-γ。用IFN-γ抗体包被磁微粒，辣根过氧化物酶标记IFN-γ抗体制备酶结合物。通过免疫反应形成抗体-抗原-抗体复合物，该复合物催化发光底物发出光子，发光强度与IFN-γ的含量成正比。本研究基于IGRA建立一种化学发光方法，并将其应用于结核病诊断。化学发光方法通过磁微粒混悬液制备，酶结合物制备，校准品制备，反应体系优化，正常参考值确定，稳定性研究建立而成。该方法应用于结核病诊断的研究主要通过与临床确诊信息对比，考核化学发光方法应用于结核病诊断的灵敏度、特异性和准确性指标，以OxfordImmunotec Ltd生产的T-SPOT.TB试剂盒作对照，考核化学发光方法应用于结核病诊断与T-SPOT.TB试剂盒的阳性符合率、阴性符合率和总符合率。建立的化学发光方法将检测时间缩短为30分钟，该化学发光方法应用于结核病诊断中，与临床确诊结果对比，灵敏度为86.19%（69/81），特异性为84.26%（589/699），准确性为84.36%（658/780）。以T-SPOT.TB试剂盒作标杆，该化学发光方法应用于结核病诊断与T-SPOT.TB试剂盒的阳性符合率为85.88%（225/262），阴性符合率为81.44%（215/264），总符合率为83.65%（440/526）。本研究通过建立化学发光方法应用于结核病的诊断，缩短了检测时间，提高了检测线性范围，更加灵敏和特异，并且对环境没有污染。为今后结核病诊断使用化学发光方法奠定基础。
[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- 纬) 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- 纬 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- 纬 was released. The magnetic particles were coated with IFN- 纬 antibody, and the IFN- 纬 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- 纬. 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.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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