自身免疫性疾病抗体检测芯片的研制

发布时间：2018-06-03 11:00

本文选题：蛋白质芯片 + 自身抗体　；参考：《山东省医学科学院》2006年硕士论文

【摘要】： 自身免疫性疾病(Autoimmune disease,AD)是指机体免疫系统对自身抗原发生免疫应答,产生自身抗体及(或)自身致敏淋巴细胞,攻击自身靶抗原细胞和组织,使其产生病理改变和功能障碍而导致的疾病。这类疾病的发病率较高,目前我国患者有一亿。国际上通用的自身免疫性疾病的诊断标准除了相应的临床表现外,主要还依据患者血清中检测到自身抗体。每种自身免疫性疾病不仅产生一种自身抗体,而经常表现的是多种自身抗体谱。目前自身抗体的检测主要采用免疫学方法,常用的有免疫印迹、免疫荧光、酶联免疫吸附实验及放射免疫测定等,在临床上,应用这些技术检测自身抗体,需要逐项去做,给临床检测带来较多的麻烦,同时试验成本也高,因此非常有必要研究开发一种可以同时检测多种自身抗体的诊断技术,1996年诞生的生物芯片技术之高通量、平行检测的优势能满足这一需要。目的:本研究对基于抗原抗体反应的蛋白芯片,进行了抗原制备以及基片、点样液和杂交条件的优化筛选,初步研制出可同时检测多种抗原特异性抗体的蛋白质芯片,既能同时检测多人份,又能检测单人份,为将来给临床提供一种高效实用的自身抗体检测技术与方法奠定基础。方法: 1.抗原的选择、制备自行制备核抗原和双链DNA(dsDNA)。 2.玻片的选择和制备及点样液的选择分别制备多聚赖氨酸、戊二醛磷酸盐溶液、戊二醛水溶液、3-氨丙基三乙氧基硅烷(APES)修饰的玻片,加上购买的3-氨基丙基三甲氧基硅烷(APMS)玻片,共五种不同修饰方法的玻片,分别以磷酸盐缓冲液、碳酸盐缓冲液、Tris缓冲液等12种液体为点样液,以人IgG为抗原,以Cy3荧光标记的羊抗人IgG为抗体制备芯片,根据检测结果优选固定效果好、信噪比高的固相基片和固定效果好的点样液。 3.蛋白芯片制备及检测条件的优化采用L9(34)正交法,Scanarray4000激光共聚扫描仪成像,Quantarray软件分析荧光强度,根据结果,筛选最佳条件。 4.蛋白芯片二抗及血清反应条件的优化采用L4(23)正交法,Scanarray4000激光共聚扫描仪成像,Quantarray软件分析荧光强度,根据结果,筛选最佳条件。 5.抗原浓度和血清稀释度梯度的确定对每一种抗原进行不同梯度稀释及血清稀释,制备芯片并检测,确定七种抗原最佳的点样浓度和血清最佳稀释度。
[Abstract]:Autoimmune disease (ADD) refers to the immune response of the body's immune system to autoantigens, the production of autoantibodies and / or autoallergenic lymphocytes, and attacks on autoantigen cells and tissues. A disease that causes pathological changes and dysfunction. The incidence of this kind of disease is relatively high, there are 100 million patients in our country at present. In addition to the clinical manifestations, the international diagnostic criteria for autoimmune diseases are mainly based on the detection of autoantibodies in patients' serum. Each autoimmune disease produces not only one autoantibody, but often multiple autoantibody profiles. At present, autoantibodies are mainly detected by immunological methods, such as immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay and radioimmunoassay. In clinical practice, the detection of autoantibodies by these techniques needs to be done one by one. It is very necessary to develop a diagnostic technology that can detect multiple autoantibodies simultaneously. The high throughput of biochip technology was born in 1996. The advantage of parallel detection can meet this need. Objective: in this study, a protein chip based on antigen-antibody reaction was prepared and optimized for antigen preparation, substrate, sample solution and hybridization, and a protein chip was developed for simultaneous detection of various antigen-specific antibodies. It can detect both multiple and single samples at the same time, which lays a foundation for the clinical application of an efficient and practical autoantibody detection technique in the future. Methods: 1. The selection of antigen, the preparation of nuclear antigen and double strand DNA dsDNA. 2. Polylysine, glutaraldehyde phosphate solution, glutaraldehyde aqueous solution, 3-aminopropyltriethoxysilane (APESs) modified glass, and 3- aminopropyl trimethoxysilane (APMS) glass were prepared by the selection and preparation of glass slide and sample solution, respectively. Five kinds of glass slides were prepared by using phosphate buffer and carbonate buffer as spot solution, human IgG as antigen and sheep anti-human IgG labeled with Cy3 as antibody. According to the detection results, the solid substrate with high SNR and the sample liquid with good fixation effect are selected. 3. The preparation and detection conditions of protein chip were optimized by L9 / 34) Quantarray software was used to analyze the fluorescence intensity of Scanarray4000 laser copolymerization scanner. According to the results, the optimum conditions were selected. 4. The optimum reaction conditions of protein chip second antibody and serum were analyzed by Quantarray software of Scanarray4000 laser copolymerization scanner. The optimum conditions were screened according to the results. 5. The antigen concentration and serum dilution gradient were determined by different gradient dilution of each antigen and serum dilution. The microarray was prepared and detected to determine the best sample concentration of seven antigens and the best dilution degree of serum.
【学位授予单位】：山东省医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392.1

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