环境污染物DBP和AFTM1单克隆抗体制备及人体内暴露水平研究
发布时间:2018-07-22 20:23
【摘要】:随着科技的进步,经济的发展,环境问题越来越凸显。2013年全国肿瘤登记中心对2010年收集到的肿瘤数据进行审核、整理和分析后发现近几年肿瘤疾病的负担日益严重,特别提到与环境相关的健康问题应作为焦点进行跟踪监测。作为导致重要疾病的因素之一,环境问题受到越来越多人的关注与重视。之前对环境污染物质检测的研究大多是针对大气、土壤、食物等外环境的监测,这些数据可以间接反映小分子环境污染物的主要暴露来源,从源头控制人类过多的暴露。直接对人体尿样、血样等生物样本中的环境污染物质进行内暴露检测能够客观的反映个体内暴露水平,避免个体差异的影响;更有效的预测、预防特定环境污染物暴露剂量对人体危害;同时可以为环境管理者在研制有害物质及致癌环境卫生学标准、提出环境中有害化学物及致癌物的可接受浓度时提供科学理论依据,进而指导国家卫生部门制定更有效的政策,提高国民的健康状态。目前针对小分子环境污染物的检测方法有仪器分析法和免疫学分析方法两种,传统的仪器分析法(HPLC、GC-MS等)不仅操作复杂,价格昂贵,通量低而且耗时长、灵敏度低,这些缺点限制了其在普通人群中的应用与普及。免疫学的方法采用抗原抗体特异性结合的原理进行检测。目前由于小分子环境污染物质完全抗原的质量低、抗体的特异性差以及方法的灵敏度低、通量低等严重的阻碍了小分子环境污染物质检测技术的发展与应用。本研究在通过细胞融合技术、高强度多次的亚克隆筛选到特异性好、效价高的单克隆抗体的基础上,建立一种灵敏度高、通量高且操作简便的检测方法,使其能够适用于大批量环境污染物邻苯二甲酸二丁酯(Dibutyl phthalate, DBP)和黄曲霉毒素M1 (Aflatoxin M1, AFTM1)内暴露样本的检测。绝大多数小分子环境污染物分子量小,只具有反应原性,而不具有免疫原性,因此针对同一小分子环境污染物需要首先合成2种不同的人工完全抗原作为免疫原和包被原,再选择制备亲和力高的特异性抗体,建立、优化检测方法后,对浙江省普通人群体内的DBP和AFTM1进行检测分析,获得该人群内暴露水平的第一手资料。一.两种小分子环境污染物人工完全抗原的制备与获得小分子环境污染物如邻苯二甲酸二丁酯(DBP)、黄曲霉毒素M1 (AFTM1),分子量分别为278.34与328,这类物质分子量小、结构简单,必须与载体蛋白结合制备人工完全抗原后才能有效的刺激动物体内产生特异性的抗体。常用的载体蛋白有牛血清白蛋白(BSA)、鸡卵血清白蛋白(OVA)、钥孔血蓝蛋白(KLH)及多聚赖氨酸(PLL)。偶联臂的应用可以使得半抗原远离大分子载体,避免小分子半抗原被载体大分子遮盖影响其免疫原性,更好的刺激免疫系统。目前应用的连接臂原形有酸酐、碳二亚胺、戊二醛等。根据半抗原的结构基团,分子中含有羧基和可羧化的半抗原可使用混合酸干法与碳二亚胺法,戊二醛法与重氮化法用于含有氨基或者可还原硝基半抗原的小分子偶联,而含有羟基的半抗原可采用琥珀酸酐和羰基二咪唑作为偶联臂。针对DBP小分子环境污染物,该研究选用BSA与OVA作为偶联载体。在4-硝基邻苯二甲酸的基础上,首先创新型的选择催化剂进行酯化合成4-硝基邻苯二甲酸二丁酯,然后选择最优的还原剂还原得到具有氨基结构的4-氨基邻苯二甲酸二丁酯,最后通过重氮化反应偶联蛋白载体获得2种人工完全抗原。DBP-BSA作为免疫小鼠的免疫抗原,DBP-OVA作为建立方法的包被抗原。优化实验条件后,该合成方法较文献中的方法产率提高,合成时间缩短。AFTM1-BSA和AFTM1-OVA的2种小分子环境污染物人工完全抗原则通过购买获得,前者作为免疫抗原,后者作为包被抗原。二.两种小分子环境污染物单克隆抗体的制备与检测方法的建立来自于同一株细胞的单克隆抗体比来自于不同只动物获得的多克隆抗体相比,具有更高的特异性、更好的均一性并且可以体内外大量制备,其来源和产量丰富等优点。因此我们采用经典的细胞融合的方法筛选得到阳性杂交瘤细胞并通过体内培养、体外纯化获得足量的单克隆抗体。继而通过优化包被抗原浓度、抗体浓度与抗体稀释液等多个条件建立最优化的间接竞争ELISA (indirect complete enzyme-linked immunosorbent assay, Ic-ELISA)检测方法。在竞争ELISA的方法中,采用制备得到的DBP-BSA抗原免疫BALB/c小鼠,经细胞融合后经4次筛选得到杂交瘤阳性细胞株,然后体内大量制备单克隆抗体。在0.25 ug/ml的抗原包被浓度下,采用0.1%明胶稀释液稀释抗体16000倍,该方法灵敏度最高:IC50= 7.34 ng/ml,线性范围为0-50 ng/ml, R2=0.994,最低检测限为0.06 ng/ml,变异系数范围为5.93-11.09%,回收率范围97.8-114.3%。同时与GC-MS传统仪器分析法相关性良好,且较文献中其它各种方法相比,具有更强的特异性、更高的灵敏度、更好的精密度和准确度。依托筛选得到的阳性杂交瘤细胞制备AFTM1单克隆抗体,优化ELISA实验后最佳包被抗原浓度为0.5 ug/ml,抗体稀释2000倍,方法的最低检测限为4 ng/L,IC50为0.36 ng/ml,可接受变异系数和回收率分别为1.788-8.678%,94.21-102.3%。该方法比其他文献检测方法相比,具有灵敏度高、特异性强等优点。根据两种小分子环境污染物的的单克隆抗体建立的ELISA方法除了特异性好、灵敏度高、操作简单以外,与仪器分析方法相比极大的提高了检测的通量,可用于大量样本中环境污染物质的检测。三.两种小分子环境污染物人体内暴露的检测与分析根据上述建立的方法,对1246个普通人群样本进行DBP和AFTM1内暴露含量的检测,检测数据采用PRISM和SPSS软件进行统计分析。DBP在人体尿样样本中的检出率为72.87%,所有检测样本的几何平均值为3 ng/ml。女性、中年组和低教育程度的人群分别较男性、老人组和高教育组人群含量高。AFTM1的检测范围为0.0004094-0.9284 ng/ml,检出率和几何平均值分别为83.01%与0.09229 ng/ml.通过性别、文化程度与年龄分析,结果显示男性、中年组和低教育人群含量较高。
[Abstract]:With the progress of science and technology, economic development and environmental problems, the tumor data collected by the National Cancer Registration Center in.2013 in 2010 are reviewed and reviewed. After sorting and analyzing, the burden of tumor disease is increasingly serious in recent years, especially the environmental related health problems should be tracked as the focal point. One of the factors of important diseases, environmental problems have attracted more and more attention and attention. Most of the previous studies on the detection of environmental pollutants are aimed at the monitoring of the atmosphere, soil, food and other external environment. These data can indirectly reflect the main source of exposure of small molecular environmental pollutants, and control excessive exposure from the source from source. Internal exposure detection of environmental pollutants in biological samples such as human urine, blood samples and other biological samples can objectively reflect the exposure level of individuals, avoid the influence of individual differences, and more effectively predict the exposure dose of specific environmental pollutants to the human body; at the same time, it can be used for environmental management to develop harmful substances and carcinogenic environment. The standard of hygiene provides scientific theoretical basis for the acceptable concentration of harmful chemicals and carcinogens in the environment, and then directs the national health department to formulate more effective policies to improve the health of the people. At present, there are two methods of instrumental analysis and immunological analysis for the detection of small molecular environmental pollutants. HPLC (GC-MS, etc.) is not only complicated, expensive, low fluxes, long time consuming and low sensitivity. These shortcomings restrict its application and popularization in the general population. The immunological method is detected by the principle of antigen antibody specific binding. The low specificity of the body and the low sensitivity of the method and the low flux have hindered the development and application of the detection technology of small molecule environmental contaminants. This study has established a high sensitivity, high flux and high flux on the basis of cell fusion technology and high intensity subclone screening to high specific and high potency monoclonal antibodies. The simple detection method can be used to detect the exposure samples of dibutyl phthalate (Dibutyl phthalate, DBP) and aflatoxin M1 (Aflatoxin M1, AFTM1) in large mass environmental pollutants. Most of the small molecular environmental pollutants have small molecular weight, only reactivity and no immunogenicity. A small molecular environmental pollutant should first synthesize 2 different artificial complete antigens as immunogen and envelope, and then select and prepare specific antibodies with high affinity, establish and optimize the detection methods, the DBP and AFTM1 in the general population of Zhejiang province were detected and analyzed, and the first hand information of the exposure level in the population was obtained. 1. Two The preparation of artificial complete antigens of small molecular environmental pollutants and the acquisition of small molecular environmental pollutants such as dibutyl phthalate (DBP) and aflatoxin M1 (AFTM1) are 278.34 and 328 respectively. The molecular weight of these substances is small and the structure is simple. It is necessary to combine with the carrier protein to prepare the artificial complete antigen to stimulate the animal body effectively. There are specific antibodies. The commonly used carrier proteins are bovine serum albumin (BSA), chicken egg serum albumin (OVA), keyhole hemocyanin (KLH) and polylysine (PLL). The application of the coupling arm can make the hapten far away from the macromolecule carrier and avoid the small molecular semi anti original carrier large molecular covering to influence its immunogenicity, and better stimulate the immunity. Phytophthora system. The origin of the connected arm is acid anhydride, carbon two imide, glutaraldehyde and so on. According to the structural groups of the hapten, the carboxyl group and the carboxylation semi antigen can be used as the mixed acid dry method and the carbon two imide method, the glutaraldehyde method and the diazotization method are used for the small molecules containing amino or reducible nitro antigen. The hydroxyl hapten can be used as the coupling arm of succinic anhydride and carbonyl two imidazole. In this study, BSA and OVA are selected as coupling carriers for DBP small molecular environmental pollutants. On the basis of 4- nitroo-phthalic acid, the first innovative selective catalyst is esterified to synthesize 4- nitroo-phthalate two butyl ester, and then the optimum is chosen. The original agent reduced the amino structure of 4- amino dibutyl phthalate. Finally, 2 kinds of artificial complete antigen.DBP-BSA were obtained by diazotization reaction coupling protein carrier as immune antigen of immunized mice. DBP-OVA was used as an antigen for the establishment of the method. The artificial complete antigen of 2 small molecular environmental pollutants that shorten the time of synthesis of.AFTM1-BSA and AFTM1-OVA is acquired by purchase. The former acts as an immune antigen, the latter is used as a clad antigen. The preparation and detection method of monoclonal antibodies to two. Two small molecular environmental pollutants from the same cell is derived from the monoclonal antibody ratio from the same cell Compared with the polyclonal antibodies obtained by animals, they have higher specificity, better homogeneity, and can be prepared in large quantities within and outside the body. Therefore, we use classical cell fusion methods to screen positive hybridoma cells and obtain sufficient monoclonal anti - monoclonal antibodies in vitro. Then the optimized indirect competitive ELISA (indirect complete enzyme-linked immunosorbent assay, Ic-ELISA) detection method was established by optimizing the concentration of antigen, antibody concentration and antibody diluent. In the competitive ELISA method, the prepared DBP-BSA antigen was immune to BALB/c mice, and the cells were fused after cell fusion. The hybridoma positive cell line was screened at 4 times, and then a large number of monoclonal antibodies were prepared in the body. Under the concentration of 0.25 ug/ml, 0.1% gelatin diluents were used to dilute the antibody 16000 times. The sensitivity of the method was the highest: IC50= 7.34 ng/ml, the linear range of 0-50 ng/ml, R2=0.994, the minimum detection limit of 0.06 ng/ml, and the range of variation coefficient of 5.93. -11.09%, the recovery range 97.8-114.3%. has a good correlation with GC-MS traditional instrument analysis, and has a stronger specificity, higher sensitivity, better precision and accuracy compared with the other methods in the literature. Based on the screened positive hybridoma cells, the AFTM1 monoclonal antibody is prepared and the optimal package after the ELISA experiment is optimized. The antigen concentration is 0.5 ug/ml, the antibody is diluted 2000 times, the minimum detection limit of the method is 4 ng/L, the IC50 is 0.36 ng/ml, the acceptable variation coefficient and the recovery rate are 1.788-8.678% respectively. Compared with the other methods of literature detection, the method has the advantages of high sensitivity and specificity, according to the monomer of two small molecular environmental pollutants. In addition to the specificity, high sensitivity and simple operation of the clone antibody, the ELISA method can greatly improve the flux of detection compared with the instrument analysis method, and can be used for the detection of environmental pollutants in a large number of samples. Three. The detection and analysis of the exposure of two small molecular environmental pollutants in human body is based on the above method, to 1246 A general population sample was tested for DBP and AFTM1 exposure, and the detection data were statistically analyzed by PRISM and SPSS software. The detection rate of.DBP in human urine samples was 72.87%. The geometric mean of all the samples was 3 ng/ml. women. The middle age group and the low education group were compared with men, the elderly group and the high education group respectively. The detection range of high.AFTM1 was 0.0004094-0.9284 ng/ml, the detection rate and the geometric mean value were 83.01% and 0.09229 ng/ml. respectively through sex, cultural degree and age analysis. The results showed that the male, the middle age group and the low education population were higher.
【学位授予单位】:浙江大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:R114
本文编号:2138466
[Abstract]:With the progress of science and technology, economic development and environmental problems, the tumor data collected by the National Cancer Registration Center in.2013 in 2010 are reviewed and reviewed. After sorting and analyzing, the burden of tumor disease is increasingly serious in recent years, especially the environmental related health problems should be tracked as the focal point. One of the factors of important diseases, environmental problems have attracted more and more attention and attention. Most of the previous studies on the detection of environmental pollutants are aimed at the monitoring of the atmosphere, soil, food and other external environment. These data can indirectly reflect the main source of exposure of small molecular environmental pollutants, and control excessive exposure from the source from source. Internal exposure detection of environmental pollutants in biological samples such as human urine, blood samples and other biological samples can objectively reflect the exposure level of individuals, avoid the influence of individual differences, and more effectively predict the exposure dose of specific environmental pollutants to the human body; at the same time, it can be used for environmental management to develop harmful substances and carcinogenic environment. The standard of hygiene provides scientific theoretical basis for the acceptable concentration of harmful chemicals and carcinogens in the environment, and then directs the national health department to formulate more effective policies to improve the health of the people. At present, there are two methods of instrumental analysis and immunological analysis for the detection of small molecular environmental pollutants. HPLC (GC-MS, etc.) is not only complicated, expensive, low fluxes, long time consuming and low sensitivity. These shortcomings restrict its application and popularization in the general population. The immunological method is detected by the principle of antigen antibody specific binding. The low specificity of the body and the low sensitivity of the method and the low flux have hindered the development and application of the detection technology of small molecule environmental contaminants. This study has established a high sensitivity, high flux and high flux on the basis of cell fusion technology and high intensity subclone screening to high specific and high potency monoclonal antibodies. The simple detection method can be used to detect the exposure samples of dibutyl phthalate (Dibutyl phthalate, DBP) and aflatoxin M1 (Aflatoxin M1, AFTM1) in large mass environmental pollutants. Most of the small molecular environmental pollutants have small molecular weight, only reactivity and no immunogenicity. A small molecular environmental pollutant should first synthesize 2 different artificial complete antigens as immunogen and envelope, and then select and prepare specific antibodies with high affinity, establish and optimize the detection methods, the DBP and AFTM1 in the general population of Zhejiang province were detected and analyzed, and the first hand information of the exposure level in the population was obtained. 1. Two The preparation of artificial complete antigens of small molecular environmental pollutants and the acquisition of small molecular environmental pollutants such as dibutyl phthalate (DBP) and aflatoxin M1 (AFTM1) are 278.34 and 328 respectively. The molecular weight of these substances is small and the structure is simple. It is necessary to combine with the carrier protein to prepare the artificial complete antigen to stimulate the animal body effectively. There are specific antibodies. The commonly used carrier proteins are bovine serum albumin (BSA), chicken egg serum albumin (OVA), keyhole hemocyanin (KLH) and polylysine (PLL). The application of the coupling arm can make the hapten far away from the macromolecule carrier and avoid the small molecular semi anti original carrier large molecular covering to influence its immunogenicity, and better stimulate the immunity. Phytophthora system. The origin of the connected arm is acid anhydride, carbon two imide, glutaraldehyde and so on. According to the structural groups of the hapten, the carboxyl group and the carboxylation semi antigen can be used as the mixed acid dry method and the carbon two imide method, the glutaraldehyde method and the diazotization method are used for the small molecules containing amino or reducible nitro antigen. The hydroxyl hapten can be used as the coupling arm of succinic anhydride and carbonyl two imidazole. In this study, BSA and OVA are selected as coupling carriers for DBP small molecular environmental pollutants. On the basis of 4- nitroo-phthalic acid, the first innovative selective catalyst is esterified to synthesize 4- nitroo-phthalate two butyl ester, and then the optimum is chosen. The original agent reduced the amino structure of 4- amino dibutyl phthalate. Finally, 2 kinds of artificial complete antigen.DBP-BSA were obtained by diazotization reaction coupling protein carrier as immune antigen of immunized mice. DBP-OVA was used as an antigen for the establishment of the method. The artificial complete antigen of 2 small molecular environmental pollutants that shorten the time of synthesis of.AFTM1-BSA and AFTM1-OVA is acquired by purchase. The former acts as an immune antigen, the latter is used as a clad antigen. The preparation and detection method of monoclonal antibodies to two. Two small molecular environmental pollutants from the same cell is derived from the monoclonal antibody ratio from the same cell Compared with the polyclonal antibodies obtained by animals, they have higher specificity, better homogeneity, and can be prepared in large quantities within and outside the body. Therefore, we use classical cell fusion methods to screen positive hybridoma cells and obtain sufficient monoclonal anti - monoclonal antibodies in vitro. Then the optimized indirect competitive ELISA (indirect complete enzyme-linked immunosorbent assay, Ic-ELISA) detection method was established by optimizing the concentration of antigen, antibody concentration and antibody diluent. In the competitive ELISA method, the prepared DBP-BSA antigen was immune to BALB/c mice, and the cells were fused after cell fusion. The hybridoma positive cell line was screened at 4 times, and then a large number of monoclonal antibodies were prepared in the body. Under the concentration of 0.25 ug/ml, 0.1% gelatin diluents were used to dilute the antibody 16000 times. The sensitivity of the method was the highest: IC50= 7.34 ng/ml, the linear range of 0-50 ng/ml, R2=0.994, the minimum detection limit of 0.06 ng/ml, and the range of variation coefficient of 5.93. -11.09%, the recovery range 97.8-114.3%. has a good correlation with GC-MS traditional instrument analysis, and has a stronger specificity, higher sensitivity, better precision and accuracy compared with the other methods in the literature. Based on the screened positive hybridoma cells, the AFTM1 monoclonal antibody is prepared and the optimal package after the ELISA experiment is optimized. The antigen concentration is 0.5 ug/ml, the antibody is diluted 2000 times, the minimum detection limit of the method is 4 ng/L, the IC50 is 0.36 ng/ml, the acceptable variation coefficient and the recovery rate are 1.788-8.678% respectively. Compared with the other methods of literature detection, the method has the advantages of high sensitivity and specificity, according to the monomer of two small molecular environmental pollutants. In addition to the specificity, high sensitivity and simple operation of the clone antibody, the ELISA method can greatly improve the flux of detection compared with the instrument analysis method, and can be used for the detection of environmental pollutants in a large number of samples. Three. The detection and analysis of the exposure of two small molecular environmental pollutants in human body is based on the above method, to 1246 A general population sample was tested for DBP and AFTM1 exposure, and the detection data were statistically analyzed by PRISM and SPSS software. The detection rate of.DBP in human urine samples was 72.87%. The geometric mean of all the samples was 3 ng/ml. women. The middle age group and the low education group were compared with men, the elderly group and the high education group respectively. The detection range of high.AFTM1 was 0.0004094-0.9284 ng/ml, the detection rate and the geometric mean value were 83.01% and 0.09229 ng/ml. respectively through sex, cultural degree and age analysis. The results showed that the male, the middle age group and the low education population were higher.
【学位授予单位】:浙江大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:R114
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