血小板糖鄂蛋白的ELISA方法的建立与血小板活化因子对脑梗死患者筛选诊断的意义

发布时间：2018-05-15 17:32

本文选题：ELISA方法 + 脑梗死　；参考：《苏州大学》2014年硕士论文

【摘要】：背景 1.酶联免疫吸附测定法(ELISA方法)是采用抗原与抗体的特异反应将待测物与酶连接，然后通过酶与底物产生颜色反应，用于定量测定。操作步骤如下：（1）将特异性抗体与固相载体连接，形成固相抗体。（2）加受检标本：使之与固相抗体接触反应一段时间，让标本中的抗原与固相载体上的抗体结合，形成固相抗原复合物。（3）加酶标抗体：使固相免疫复合物上的抗原与酶标抗体结合。（4）加底物：夹心式复合物中的酶催化底物成为有色产物。根据颜色反应的程度进行该抗原的定性或定量。该法具有特异性强，且操作简便，灵敏度和重复性较好等优点。 2.脑血栓是缺血性脑血管病中最常见的一种.它是由于供应脑部的动脉内有血栓形成,造成动脉管腔狭窄或完全闭塞,使其供血区局部脑组织缺血、缺氧、坏死而引起的神经功能障碍。俗称中风，脑血栓形成原因是脑血管内的动脉粥样硬化斑块使得血管狭窄,表面粗糙不平,而后斑块破裂出血，激活体内的血液凝固系统形成血栓，血液凝固过程血小板释放多种因子。急性脑血栓形成的发病涉及三个因素：血管壁结构完整性破坏，止血、凝血、纤溶系统的失平衡和血流状态的改变，三种因素相互作用，共同构成血栓形成的基本条件。近些年来，通过对脑血栓形成发病机制的深入研究，发现很多分子标志物在血栓形成过程中发挥着的重要作用，且也能够反映上述三种因素在血栓形血栓前状态．并对血栓形成后不同阶段的干预提供依据。目的 1.建立血小板糖鄂蛋白ELISA方法，并且用于临床检测。 2. SGC，P选择素，GP1bɑ对脑梗死患者筛选和诊断的意义。方法 1．选用2株互不干扰的单克隆抗体，建立双抗夹心，及选用抗体AN51做包被抗体，选SZ-2做标记抗体，用biotin标记SZ-2。AN51包被在96板孔上，再加入脑梗死患者样本（含GC）与固相载体反应，，然后再加入另一种biotin标记的抗体SZ-2，最后再与底物氧化还原反应而成颜色，测其35例脑梗死患者OD值。 2.用SZ-2抗体抗GP1bɑ再用流式检测35个脑血栓患者的GP1bɑ表达和用SZ-51抗体抗P选择素再用流式检测25个脑梗塞患者P选择素的表达。结果 1. SGcELISA测量范围0.15－5.25ug／ml,灵敏度：50ug／l，精密度：批内低、中、高值变异系数分别为8.25％、7.35％和3.99％(n=6)，批间分别为9．55％、8.82％和5.48％(n=6)；准确度：平均回收率为101.88%。35例脑梗死血清中的SGC浓度：健康对照组SGC浓度为2.04±0.46明显低于脑梗死患者的SGC浓度为;3.4±0.34ug／ml。两组间比较差异具有显著性(P 0.05)。 2.脑血栓组血小板CD62P(12.6±1.9%）阳性百分率显著高于对照组(6.46±1.50%），脑血栓血小板GP1bɑ(13.8±2.7)%阳性百分率显著低于对照组（24.31±6.5%）。两组间比较差异具有显著性(P 0.05)。结论： 1.建立血小板糖鄂蛋白ELISA方法 2. SGC，P选择素，GP1bɑ对脑梗死患者筛选和诊断的意义。
[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 卤0.46 significantly lower than that of patients with cerebral infarction of 3.4 卤0.34ug.ml. There was significant difference between the two groups (P 0.05). 2. The positive rate of platelet CD62P(12.6 卤1.9% in cerebral thrombosis group was significantly higher than that in control group (6.46 卤1.50), and the positive rate of platelet GP1b in cerebral thrombosis group was significantly lower than that in control group (24.31 卤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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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