自身抗体免疫层析检测方法与重金属铬离子纳米ELISA的建立

发布时间：2018-03-16 01:11

本文选题：自身免疫性疾病　切入点：自身抗体　出处：《暨南大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:建立一种高效、快速(5~8 min)、简便的胶体金免疫层析试纸条应用于临床检测血清中自身抗体,用来辅助诊断自身免疫性疾病(Autoimmune diseases,AID),实现检测结果信息快速、及时的反馈。方法:抗SSA/SSB自身抗体免疫层析检测技术的建立,是利用金黄色葡萄球菌A蛋白(SPA)标记于金纳米颗粒(Gold nanoparticles,Au NPs)上,并将金标SPA喷涂在结合垫上,抗原(SSA/SSB)喷涂在硝酸纤维素膜(NC膜)上特定位置作为检测线(Test line,T line),山羊抗人的多克隆抗体喷涂在NC膜的另一特定位置作为校准线(Calibration line,C line);对试纸条的样品垫、结合垫、NC膜、血清样品稀释缓冲液以及稀释倍数等方面的条件进行综合优化并组装试纸条。利用上述方法建立的胶体金免疫层析试纸条检测血清样本并与目前医院临床上常用的检测方法进行检测结果符合率的比较。结果:本实验建立的抗SSA抗体胶体金免疫层析试纸条与目前国内临床常用的LIA方法检测血清样本的结果总符合率为95.12%;本实验建立的抗SSB抗体胶体金免疫层析试纸条与目前临床常用的LIA方法检测血清样本的结果总符合率为97.82%。结论:本研究建立检测抗SSA抗体、抗SSB抗体的胶体金免疫层析试纸条检测临床血清样本与目前国内临床上使用的主要方法LIA、ELISA的检测结果均有较高的符合率,该方法操作简便,检测结果可在(5~8 min)内完成,有良好的市场应用前景。目的:建立一种新的、可裸眼观察检测结果的等离子纳米传感器,该方法检测结果判读简单,无需昂贵的仪器,可适用于医疗资源匮乏的地区。方法:利用过氧化氢能够腐蚀三角银纳米片,使得三角银纳米片的尺寸大小和形状发生改变,局部等离子共振(LSPR)发生改变从而表现出特有的光学特征(由蓝色变紫色)。根据这一现象,本研究建立了一种等离子纳米传感器去检测实际水样中的三价铬离子Cr(III)。该方法主要是在竞争ELISA的基础上(过氧化氢酶标记二抗),利用过氧化氢和三角银作为底物显色系统,过氧化氢酶消耗过氧化氢来调控体系中剩余过氧化氢的浓度,进而调控三角银纳米片受腐蚀的程度。三角银的这种LSPR变化强度(颜色和吸收光谱)与铬的浓度呈正相关,我们对该方法进行可行性验证、灵敏度和特异性评价并用该方法检测实际水样中的加标Cr(III)的浓度,检测结果与ICP-MS的检测结果进行比较。结果:本研究建立的等离子传感器检测Cr(III)可裸眼检测的灵敏度达到6.25 ng/m L。用银纳米片的LSPR位移分析时,该方法的线性范围为3.13 ng/m L-50 ng/m L,最低检测限为3.13 ng/m L,并且显示了良好的特异性。结论:本研究建立的检测Cr(III)的等离子纳米传感器,可以用于医疗资源匮乏的地区,无需使用昂贵的仪器也能对实际水样中的重金属浓度做初步检测。
[Abstract]:Objective: to establish a highly efficient, rapid and rapid colloidal gold immunochromatographic test strip for clinical detection of autoimmune diseases autoimmune diseases (autoimmune diseases) AIDD, and to realize the rapid information of the detection results, and to establish a simple and convenient colloidal gold immunochromatographic test strip for the clinical detection of autoimmune diseases (autoimmune diseases) and autoimmune diseases (AIDD). Methods: the establishment of anti SSA/SSB autoantibody immunochromatographic technique was to use staphylococcus aureus protein A protein (spa) to label gold nanoparticles Gold nanoparticleson au NPs and spray gold labeled SPA on the binding pad. Antigenic SSA-SSBs sprayed on the nitrocellulose membrane / NC membrane) as the test line test line, goat anti-human polyclonal antibody sprayed at another specific location of the NC membrane as calibration line Calibration line line; for the sample pad of the test strip, combined with the pad for the NC film, The conditions of dilution buffer and dilution multiple of serum sample were optimized and the test strip was assembled. The colloidal gold immunochromatographic strip was used to detect the serum sample and it was compared with the current clinical practice in hospital. Results: the total coincidence rate of the test strip established in this experiment with that of the LIA method commonly used in China is 95.12%. The total coincidence rate of the gold immunochromatographic strip established by the experiment for the detection of serum samples by LIA method is 97.82.Conclusion: this study established the method for detection of anti SSA antibody. The detection of serum samples by colloidal gold immunochromatographic test strip of anti-#en0# antibody has a high coincidence rate with that of the main clinical method used in China at present. The method is simple and easy to operate, and the result can be completed within 58 mins. Objective: to establish a new plasma nanosensor which can be observed by naked eyes. Methods: hydrogen peroxide can be used to corrode triangular silver nanoparticles and change their size and shape. Local plasmon resonance (LSPRA) changes to exhibit unique optical characteristics (from blue to purple). According to this phenomenon, In this study, a plasma nanosensor was developed to detect trivalent chromium ions in real water samples. The method was mainly based on competitive ELISA (catalase labeled second antibody, using hydrogen peroxide and silver triangle as substrate color system). Catalase consumes hydrogen peroxide to regulate the concentration of residual hydrogen peroxide in the system, and then to regulate the degree of corrosion of triangular silver nanoparticles. The intensity of LSPR change (color and absorption spectrum) of triangular silver is positively correlated with the concentration of chromium. We verified the feasibility of the method, evaluated the sensitivity and specificity of the method, and used this method to detect the concentration of labeled Cr-III in real water samples. The results were compared with those of ICP-MS. Results: the sensitivity of the plasma sensor developed in this study was 6.25 ng/m / L. when using the LSPR displacement analysis of silver nanoparticles, The linear range of this method is 3.13 ng/m L-50 ng/m / L, and the lowest detection limit is 3.13 ng/m / L, and it shows good specificity. Conclusion: the plasma nanosensor developed in this study can be used in the areas with scarce medical resources. The concentrations of heavy metals in real water samples can be preliminarily detected without the use of expensive instruments.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.6

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