本文選題：ELISA方法 + 腦梗死　； 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：背景 1.酶聯(lián)免疫吸附測(cè)定法(ELISA方法)是采用抗原與抗體的特異反應(yīng)將待測(cè)物與酶連接，然后通過酶與底物產(chǎn)生顏色反應(yīng)，用于定量測(cè)定。操作步驟如下：（1）將特異性抗體與固相載體連接，形成固相抗體。（2）加受檢標(biāo)本：使之與固相抗體接觸反應(yīng)一段時(shí)間，讓標(biāo)本中的抗原與固相載體上的抗體結(jié)合，形成固相抗原復(fù)合物。（3）加酶標(biāo)抗體：使固相免疫復(fù)合物上的抗原與酶標(biāo)抗體結(jié)合。（4）加底物：夾心式復(fù)合物中的酶催化底物成為有色產(chǎn)物。根據(jù)顏色反應(yīng)的程度進(jìn)行該抗原的定性或定量。該法具有特異性強(qiáng)，且操作簡(jiǎn)便，靈敏度和重復(fù)性較好等優(yōu)點(diǎn)。 2.腦血栓是缺血性腦血管病中最常見的一種.它是由于供應(yīng)腦部的動(dòng)脈內(nèi)有血栓形成,造成動(dòng)脈管腔狹窄或完全閉塞,使其供血區(qū)局部腦組織缺血、缺氧、壞死而引起的神經(jīng)功能障礙。俗稱中風(fēng)，腦血栓形成原因是腦血管內(nèi)的動(dòng)脈粥樣硬化斑塊使得血管狹窄,表面粗糙不平,而后斑塊破裂出血，激活體內(nèi)的血液凝固系統(tǒng)形成血栓，血液凝固過程血小板釋放多種因子。急性腦血栓形成的發(fā)病涉及三個(gè)因素：血管壁結(jié)構(gòu)完整性破壞，止血、凝血、纖溶系統(tǒng)的失平衡和血流狀態(tài)的改變，三種因素相互作用，共同構(gòu)成血栓形成的基本條件。近些年來，通過對(duì)腦血栓形成發(fā)病機(jī)制的深入研究，發(fā)現(xiàn)很多分子標(biāo)志物在血栓形成過程中發(fā)揮著的重要作用，且也能夠反映上述三種因素在血栓形血栓前狀態(tài)．并對(duì)血栓形成后不同階段的干預(yù)提供依據(jù)。 目的 1.建立血小板糖鄂蛋白ELISA方法，并且用于臨床檢測(cè)。 2. SGC，P選擇素，GP1bɑ對(duì)腦梗死患者篩選和診斷的意義。 方法 1．選用2株互不干擾的單克隆抗體，建立雙抗夾心，及選用抗體AN51做包被抗體，選SZ-2做標(biāo)記抗體，用biotin標(biāo)記SZ-2。AN51包被在96板孔上，再加入腦梗死患者樣本（含GC）與固相載體反應(yīng)，，然后再加入另一種biotin標(biāo)記的抗體SZ-2，最后再與底物氧化還原反應(yīng)而成顏色，測(cè)其35例腦梗死患者OD值。 2.用SZ-2抗體抗GP1bɑ再用流式檢測(cè)35個(gè)腦血栓患者的GP1bɑ表達(dá)和用SZ-51抗體抗P選擇素再用流式檢測(cè)25個(gè)腦梗塞患者P選擇素的表達(dá)。 結(jié)果 1. SGcELISA測(cè)量范圍0.15－5.25ug／ml,靈敏度：50ug／l，精密度：批內(nèi)低、中、高值變異系數(shù)分別為8.25％、7.35％和3.99％(n=6)，批間分別為9．55％、8.82％和5.48％(n=6)；準(zhǔn)確度：平均回收率為101.88%。35例腦梗死血清中的SGC濃度：健康對(duì)照組SGC濃度為2.04±0.46明顯低于腦梗死患者的SGC濃度為;3.4±0.34ug／ml。兩組間比較差異具有顯著性(P 0.05)。 2.腦血栓組血小板CD62P(12.6±1.9%）陽性百分率顯著高于對(duì)照組(6.46±1.50%），腦血栓血小板GP1bɑ(13.8±2.7)%陽性百分率顯著低于對(duì)照組（24.31±6.5%）。兩組間比較差異具有顯著性(P 0.05)。 結(jié)論： 1.建立血小板糖鄂蛋白ELISA方法 2. SGC，P選擇素，GP1bɑ對(duì)腦梗死患者篩選和診斷的意義。
[Abstract]:Background 1. Enzyme-linked immunosorbent assay (Elisa) is a specific reaction of antigen and antibody to connect the tested substance to the enzyme, and then to produce a color reaction between the enzyme and the substrate for quantitative determination. The operation steps are as follows: (1) the specific antibody is connected with the solid carrier to form a solid phase antibody. 2) and the tested specimen is added: contact with the solid phase antibody for a period of time, so that the antigen in the sample binds to the antibody on the solid phase carrier. To form solid phase antigen complex. 3) Enzyme-labeled antibody: binding antigen on solid immune complex to enzyme labeled antibody. 4) substrate: enzyme catalyzed substrate in sandwich complex becomes colored product. The antigen is qualitatively or quantitatively determined according to the degree of color reaction. The method has the advantages of high specificity, simple operation, good sensitivity and repeatability. 2. Cerebral thrombosis is the most common type of ischemic cerebrovascular disease. It is caused by thrombosis in the arteries supplying the brain, resulting in stenosis or complete occlusion of the arterial lumen, which results in ischemia, hypoxia and necrosis of the regional brain tissue in the blood supply area. Commonly known as stroke, cerebral thrombosis is caused by atherosclerotic plaques in the cerebral vessels that cause stenosis, rough surfaces, and then plaque rupture and bleeding, which activate the blood coagulation system in the body to form thrombosis. Platelets release multiple factors during blood coagulation. The pathogenesis of acute cerebral thrombosis involves three factors: the destruction of vascular wall structural integrity, hemostasis, coagulation, the imbalance of fibrinolytic system and the change of blood flow state. The three factors interact together to form the basic conditions of thrombosis. In recent years, through the in-depth study of the pathogenesis of cerebral thrombosis, it is found that many molecular markers play an important role in the process of thrombosis, and can also reflect the above three factors in the pre-thrombotic state. And to provide the basis for intervention in different stages after thrombosis. Purpose 1. To establish a ELISA assay for platelet glycoprotein and to use it in clinical detection. 2. The significance of SGC- P selectin GP1b in screening and diagnosis of cerebral infarction patients. Method 1. Two non-interference monoclonal antibodies were used to establish double antibody sandwich, AN51 was used as coating antibody, SZ-2 was used as labeled antibody, biotin was used to label SZ-2.AN51 on 96 plate holes, and then the samples of patients with cerebral infarction (including GCCs) were added to react with solid phase carrier. Then another biotin labeled antibody SZ-2 was added and then reacted with the substrate redox reaction to determine the OD value of 35 patients with cerebral infarction. 2. The expression of GP1b in 35 patients with cerebral thrombosis and 25 patients with cerebral infarction were detected by flow cytometry with anti GP1b antibody of SZ-2 and anti P selectin with anti P selectin of SZ-51 antibody. Result 1. SGcELISA measurement range 0.15-5.25ugrmlsensitivity: 50ugrl, precision: low, medium, The high coefficient of variation was 8.257.35% and 3.99%, respectively, and the interbatches were 9.55, 8.82% and 5.48%, respectively. The accuracy: the average recovery rate was 101.88.35 cases of cerebral infarction serum SGC concentration: the SGC concentration of healthy control group was 2.04 鹵0.46 significantly lower than that of patients with cerebral infarction of 3.4 鹵0.34ug.ml. There was significant difference between the two groups (P 0.05). 2. The positive rate of platelet CD62P(12.6 鹵1.9% in cerebral thrombosis group was significantly higher than that in control group (6.46 鹵1.50), and the positive rate of platelet GP1b in cerebral thrombosis group was significantly lower than that in control group (24.31 鹵6.5%). There was significant difference between the two groups (P 0.05). Conclusion: 1. Establishment of ELISA method for platelet glycoprotein 2. The significance of SGC- P selectin GP1b in screening and diagnosis of cerebral infarction patients.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.3

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