發(fā)布時間：2018-06-01 01:24

  本文選題：甲胎蛋白 + 定量分析��； 參考：《北京化工大學(xué)》2015年碩士論文

【摘要】：血清中甲胎蛋白(AFP)的準(zhǔn)確測量對于癌癥的臨床診斷和治療具有重要意義。電感耦合等離子體質(zhì)譜法(ICP-MS)由于靈敏度高、檢出限低、多元素同時檢測等優(yōu)點(diǎn),近年來在蛋白質(zhì)及典型疾病標(biāo)志物的測量中逐漸發(fā)揮出重要作用。通過稀土元素標(biāo)記蛋白質(zhì)再進(jìn)行定量的方法是一種高靈敏度、低檢出限的定量方法,本文探討了兩種高靈敏度以ICP-MS作為測量工具的蛋白質(zhì)定量方法,通過條件優(yōu)化,得出最優(yōu)條件,用蛋白質(zhì)標(biāo)準(zhǔn)品作為模板樣品進(jìn)行定量分析,根據(jù)兩種方法的檢出限來探討方法用于血清中低豐度的甲胎蛋白定量研究的可能性。本文研究的兩種方法分別為：1、通過利用DTPAA、DOTA等雙功能試劑直接在目標(biāo)蛋白質(zhì)分子中標(biāo)記上背景低、靈敏度高的稀土元素銪,再經(jīng)過一維常規(guī)液相色譜分離技術(shù)(1D-HPLC)或納升級二維液相色譜分離技術(shù)(nano 2D-HPLC)、以及凝膠電泳分離技術(shù)分離后用ICP-MS或LA-ICP-MS進(jìn)行定量分析。2、充分利用了抗原抗體免疫反應(yīng)的特異性。首先將稀土元素用雙功能螯合試劑標(biāo)記在抗體上,再通過抗原抗體的特異性免疫反應(yīng),使蛋白質(zhì)上間接帶上標(biāo)記的稀土元素,隨后通過酸性溶液解離后溶液進(jìn)樣或者直接采用激光燒蝕(LA)固體進(jìn)樣引入到ICP-MS中進(jìn)行定量分析,其中,討論了醋酸、醇類、EDTA等有機(jī)基體改進(jìn)劑的增強(qiáng)作用機(jī)理,最后成功用于血清中低豐度蛋白質(zhì)的定量,降低了檢出限(0.57 μ g·L-1)。另外,本文還通過基于ICP-MS的方法建立了免疫復(fù)合物中甲胎蛋白和標(biāo)記元素之間的結(jié)合關(guān)系,提出了該結(jié)合關(guān)系用于同位素稀釋法定量分析的可能性,同時,為目前臨床上使用的甲胎蛋白試劑盒提出了一種節(jié)約試劑盒成本和簡化試劑盒操作過程的思路。通過對高靈敏度基于ICP-MS的稀土元素標(biāo)記蛋白質(zhì)并對低豐度蛋白質(zhì)的定量方法進(jìn)行了探索,發(fā)現(xiàn)直接標(biāo)記的方法和傳統(tǒng)根據(jù)蛋白質(zhì)自身含有的元素定量的方法相比,檢出限更低,但是由于不具有特異性,對分離要求高等缺點(diǎn)不適合用于復(fù)雜基體中低豐度蛋白質(zhì)的定量。最后,借助抗原抗體的免疫反應(yīng)的特異性,使低豐度的甲胎蛋白標(biāo)記上特有的稀土元素,隨后用具有增強(qiáng)效果的5%HAc進(jìn)行解離,并引入至ICP-MS中進(jìn)行測量,該方法已成功應(yīng)用于血清中甲胎蛋白的定量,線性范圍為1～600 μ g·L-1,檢出限為0.57 μ g·L-1,測量結(jié)果的相對標(biāo)準(zhǔn)偏差(RSD)低于10%,采用基體改進(jìn)ICP-MS測量人血清中AFP的含量與TRFIA測量結(jié)果一致,且精密度要優(yōu)于TRFIA的測量結(jié)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.
【學(xué)位授予單位】：北京化工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.11;O657.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王菡;楊江民;;甲胎蛋白含量測定的臨床意義[J];青海醫(yī)藥雜志;2011年11期

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本文編號：1962293