无机质谱联用技术对血清中低丰度蛋白质的定量研究

发布时间：2018-06-01 01:24

本文选题：甲胎蛋白 + 定量分析　；参考：《北京化工大学》2015年硕士论文

【摘要】：血清中甲胎蛋白(AFP)的准确测量对于癌症的临床诊断和治疗具有重要意义。电感耦合等离子体质谱法(ICP-MS)由于灵敏度高、检出限低、多元素同时检测等优点,近年来在蛋白质及典型疾病标志物的测量中逐渐发挥出重要作用。通过稀土元素标记蛋白质再进行定量的方法是一种高灵敏度、低检出限的定量方法,本文探讨了两种高灵敏度以ICP-MS作为测量工具的蛋白质定量方法,通过条件优化,得出最优条件,用蛋白质标准品作为模板样品进行定量分析,根据两种方法的检出限来探讨方法用于血清中低丰度的甲胎蛋白定量研究的可能性。本文研究的两种方法分别为：1、通过利用DTPAA、DOTA等双功能试剂直接在目标蛋白质分子中标记上背景低、灵敏度高的稀土元素铕,再经过一维常规液相色谱分离技术(1D-HPLC)或纳升级二维液相色谱分离技术(nano 2D-HPLC)、以及凝胶电泳分离技术分离后用ICP-MS或LA-ICP-MS进行定量分析。2、充分利用了抗原抗体免疫反应的特异性。首先将稀土元素用双功能螯合试剂标记在抗体上,再通过抗原抗体的特异性免疫反应,使蛋白质上间接带上标记的稀土元素,随后通过酸性溶液解离后溶液进样或者直接采用激光烧蚀(LA)固体进样引入到ICP-MS中进行定量分析,其中,讨论了醋酸、醇类、EDTA等有机基体改进剂的增强作用机理,最后成功用于血清中低丰度蛋白质的定量,降低了检出限(0.57 μ g·L-1)。另外,本文还通过基于ICP-MS的方法建立了免疫复合物中甲胎蛋白和标记元素之间的结合关系,提出了该结合关系用于同位素稀释法定量分析的可能性,同时,为目前临床上使用的甲胎蛋白试剂盒提出了一种节约试剂盒成本和简化试剂盒操作过程的思路。通过对高灵敏度基于ICP-MS的稀土元素标记蛋白质并对低丰度蛋白质的定量方法进行了探索,发现直接标记的方法和传统根据蛋白质自身含有的元素定量的方法相比,检出限更低,但是由于不具有特异性,对分离要求高等缺点不适合用于复杂基体中低丰度蛋白质的定量。最后,借助抗原抗体的免疫反应的特异性,使低丰度的甲胎蛋白标记上特有的稀土元素,随后用具有增强效果的5%HAc进行解离,并引入至ICP-MS中进行测量,该方法已成功应用于血清中甲胎蛋白的定量,线性范围为1～600 μ g·L-1,检出限为0.57 μ g·L-1,测量结果的相对标准偏差(RSD)低于10%,采用基体改进ICP-MS测量人血清中AFP的含量与TRFIA测量结果一致,且精密度要优于TRFIA的测量结果。
[Abstract]:The accurate measurement of AFP in serum is of great significance for the clinical diagnosis and treatment of cancer. Inductively coupled plasma mass spectrometry (ICP-MS) has played an important role in the measurement of proteins and typical disease markers in recent years due to its high sensitivity, low detection limit and simultaneous detection of multiple elements. The method of requantifying protein labeled with rare earth elements is a high sensitivity and low detection limit quantitative method. This paper discusses two methods of protein quantification with high sensitivity using ICP-MS as a measuring tool and optimizes the conditions. The optimum conditions were obtained and the protein standard was used as template sample for quantitative analysis. According to the detection limit of the two methods, the possibility of using the two methods for quantitative analysis of AFP with low abundance in serum was discussed. The two methods studied in this paper are: 1. By using DTPAA DOTA and other bifunctional reagents, europium, a rare earth element with low background and high sensitivity, is labeled directly in the target protein molecule. Then 1D-HPLC or nano-2D-HPLCX were separated by one-dimensional conventional liquid chromatography, and then analyzed by ICP-MS or LA-ICP-MS after separation by gel electrophoresis, which made full use of the specificity of antigen-antibody immune reaction. First, the rare earth elements were labeled on the antibody with bifunctional chelating reagent, and then the protein was indirectly labeled with the rare earth element through the specific immune reaction of antigen and antibody. After dissociation of acid solution, the solution was injected or the solid sample was introduced into ICP-MS for quantitative analysis. The enhancement mechanism of organic matrix modifiers such as acetic acid and alcohol were discussed. Finally, it was successfully applied to the quantification of low abundance protein in serum, and the detection limit was reduced by 0.57 渭 g / L ~ (-1). In addition, the binding relationship between alpha-fetoprotein and labeled elements in immune complexes was established based on ICP-MS, and the possibility of using the binding relationship for quantitative analysis by isotope dilution method was put forward. This paper presents a method to save the cost of the kit and simplify the operation of the kit for the current clinical use of alpha-fetoprotein kit. In this paper, the high sensitivity ICP-MS based rare earth element labeling method and the quantitative method of low abundance protein were explored. It was found that the detection limit of the direct labeling method was lower than that of the traditional quantitative method based on the elements contained in the protein itself. However, it is not suitable for the quantification of low abundance protein in complex matrix because of its unspecificity and high separation requirement. Finally, with the help of the specificity of the immunological reaction of antigen and antibody, the low abundance alpha-fetoprotein was labeled with specific rare earth elements, then dissociated with the enhanced 5%HAc, and then was introduced into the ICP-MS for measurement. The method has been successfully applied to the determination of alpha-fetoprotein in serum. The linear range is 1 ~ 600 渭 g / L ~ (-1), the detection limit is 0.57 渭 g / L ~ (-1). The relative standard deviation (RSD) of the measured results is lower than 10 ~ (10). The content of AFP in human serum measured by matrix modified ICP-MS is consistent with that of TRFIA. The precision is better than that of TRFIA.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.11;O657.63

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