發(fā)布時(shí)間：2018-06-03 11:00

  本文選題：蛋白質(zhì)芯片 + 自身抗體��； 參考：《山東省醫(yī)學(xué)科學(xué)院》2006年碩士論文

【摘要】： 自身免疫性疾病(Autoimmune disease,AD)是指機(jī)體免疫系統(tǒng)對(duì)自身抗原發(fā)生免疫應(yīng)答,產(chǎn)生自身抗體及(或)自身致敏淋巴細(xì)胞,攻擊自身靶抗原細(xì)胞和組織,使其產(chǎn)生病理改變和功能障礙而導(dǎo)致的疾病。這類疾病的發(fā)病率較高,目前我國(guó)患者有一億。國(guó)際上通用的自身免疫性疾病的診斷標(biāo)準(zhǔn)除了相應(yīng)的臨床表現(xiàn)外,主要還依據(jù)患者血清中檢測(cè)到自身抗體。每種自身免疫性疾病不僅產(chǎn)生一種自身抗體,而經(jīng)常表現(xiàn)的是多種自身抗體譜。目前自身抗體的檢測(cè)主要采用免疫學(xué)方法,常用的有免疫印跡、免疫熒光、酶聯(lián)免疫吸附實(shí)驗(yàn)及放射免疫測(cè)定等,在臨床上,應(yīng)用這些技術(shù)檢測(cè)自身抗體,需要逐項(xiàng)去做,給臨床檢測(cè)帶來較多的麻煩,同時(shí)試驗(yàn)成本也高,因此非常有必要研究開發(fā)一種可以同時(shí)檢測(cè)多種自身抗體的診斷技術(shù),1996年誕生的生物芯片技術(shù)之高通量、平行檢測(cè)的優(yōu)勢(shì)能滿足這一需要。 目的:本研究對(duì)基于抗原抗體反應(yīng)的蛋白芯片,進(jìn)行了抗原制備以及基片、點(diǎn)樣液和雜交條件的優(yōu)化篩選,初步研制出可同時(shí)檢測(cè)多種抗原特異性抗體的蛋白質(zhì)芯片,既能同時(shí)檢測(cè)多人份,又能檢測(cè)單人份,為將來給臨床提供一種高效實(shí)用的自身抗體檢測(cè)技術(shù)與方法奠定基礎(chǔ)。 方法: 1.抗原的選擇、制備自行制備核抗原和雙鏈DNA(dsDNA)。 2.玻片的選擇和制備及點(diǎn)樣液的選擇分別制備多聚賴氨酸、戊二醛磷酸鹽溶液、戊二醛水溶液、3-氨丙基三乙氧基硅烷(APES)修飾的玻片,加上購(gòu)買的3-氨基丙基三甲氧基硅烷(APMS)玻片,共五種不同修飾方法的玻片,分別以磷酸鹽緩沖液、碳酸鹽緩沖液、Tris緩沖液等12種液體為點(diǎn)樣液,以人IgG為抗原,以Cy3熒光標(biāo)記的羊抗人IgG為抗體制備芯片,根據(jù)檢測(cè)結(jié)果優(yōu)選固定效果好、信噪比高的固相基片和固定效果好的點(diǎn)樣液。 3.蛋白芯片制備及檢測(cè)條件的優(yōu)化采用L9(34)正交法,Scanarray4000激光共聚掃描儀成像,Quantarray軟件分析熒光強(qiáng)度,根據(jù)結(jié)果,篩選最佳條件。 4.蛋白芯片二抗及血清反應(yīng)條件的優(yōu)化采用L4(23)正交法,Scanarray4000激光共聚掃描儀成像,Quantarray軟件分析熒光強(qiáng)度,根據(jù)結(jié)果,篩選最佳條件。 5.抗原濃度和血清稀釋度梯度的確定對(duì)每一種抗原進(jìn)行不同梯度稀釋及血清稀釋,制備芯片并檢測(cè),確定七種抗原最佳的點(diǎn)樣濃度和血清最佳稀釋度。
[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.
【學(xué)位授予單位】：山東省醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392.1

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