環(huán)境污染物DBP和AFTM1單克隆抗體制備及人體內(nèi)暴露水平研究
發(fā)布時間：2018-07-22 20:23
【摘要】：隨著科技的進(jìn)步,經(jīng)濟(jì)的發(fā)展,環(huán)境問題越來越凸顯。2013年全國腫瘤登記中心對2010年收集到的腫瘤數(shù)據(jù)進(jìn)行審核、整理和分析后發(fā)現(xiàn)近幾年腫瘤疾病的負(fù)擔(dān)日益嚴(yán)重,特別提到與環(huán)境相關(guān)的健康問題應(yīng)作為焦點(diǎn)進(jìn)行跟蹤監(jiān)測。作為導(dǎo)致重要疾病的因素之一,環(huán)境問題受到越來越多人的關(guān)注與重視。之前對環(huán)境污染物質(zhì)檢測的研究大多是針對大氣、土壤、食物等外環(huán)境的監(jiān)測,這些數(shù)據(jù)可以間接反映小分子環(huán)境污染物的主要暴露來源,從源頭控制人類過多的暴露。直接對人體尿樣、血樣等生物樣本中的環(huán)境污染物質(zhì)進(jìn)行內(nèi)暴露檢測能夠客觀的反映個體內(nèi)暴露水平,避免個體差異的影響；更有效的預(yù)測、預(yù)防特定環(huán)境污染物暴露劑量對人體危害；同時可以為環(huán)境管理者在研制有害物質(zhì)及致癌環(huán)境衛(wèi)生學(xué)標(biāo)準(zhǔn)、提出環(huán)境中有害化學(xué)物及致癌物的可接受濃度時提供科學(xué)理論依據(jù),進(jìn)而指導(dǎo)國家衛(wèi)生部門制定更有效的政策,提高國民的健康狀態(tài)。目前針對小分子環(huán)境污染物的檢測方法有儀器分析法和免疫學(xué)分析方法兩種,傳統(tǒng)的儀器分析法(HPLC、GC-MS等)不僅操作復(fù)雜,價格昂貴,通量低而且耗時長、靈敏度低,這些缺點(diǎn)限制了其在普通人群中的應(yīng)用與普及。免疫學(xué)的方法采用抗原抗體特異性結(jié)合的原理進(jìn)行檢測。目前由于小分子環(huán)境污染物質(zhì)完全抗原的質(zhì)量低、抗體的特異性差以及方法的靈敏度低、通量低等嚴(yán)重的阻礙了小分子環(huán)境污染物質(zhì)檢測技術(shù)的發(fā)展與應(yīng)用。本研究在通過細(xì)胞融合技術(shù)、高強(qiáng)度多次的亞克隆篩選到特異性好、效價高的單克隆抗體的基礎(chǔ)上,建立一種靈敏度高、通量高且操作簡便的檢測方法,使其能夠適用于大批量環(huán)境污染物鄰苯二甲酸二丁酯(Dibutyl phthalate, DBP)和黃曲霉毒素M1 (Aflatoxin M1, AFTM1)內(nèi)暴露樣本的檢測。絕大多數(shù)小分子環(huán)境污染物分子量小,只具有反應(yīng)原性,而不具有免疫原性,因此針對同一小分子環(huán)境污染物需要首先合成2種不同的人工完全抗原作為免疫原和包被原,再選擇制備親和力高的特異性抗體,建立、優(yōu)化檢測方法后,對浙江省普通人群體內(nèi)的DBP和AFTM1進(jìn)行檢測分析,獲得該人群內(nèi)暴露水平的第一手資料。一.兩種小分子環(huán)境污染物人工完全抗原的制備與獲得小分子環(huán)境污染物如鄰苯二甲酸二丁酯(DBP)、黃曲霉毒素M1 (AFTM1),分子量分別為278.34與328,這類物質(zhì)分子量小、結(jié)構(gòu)簡單,必須與載體蛋白結(jié)合制備人工完全抗原后才能有效的刺激動物體內(nèi)產(chǎn)生特異性的抗體。常用的載體蛋白有牛血清白蛋白(BSA)、雞卵血清白蛋白(OVA)、鑰孔血藍(lán)蛋白(KLH)及多聚賴氨酸(PLL)。偶聯(lián)臂的應(yīng)用可以使得半抗原遠(yuǎn)離大分子載體,避免小分子半抗原被載體大分子遮蓋影響其免疫原性,更好的刺激免疫系統(tǒng)。目前應(yīng)用的連接臂原形有酸酐、碳二亞胺、戊二醛等。根據(jù)半抗原的結(jié)構(gòu)基團(tuán),分子中含有羧基和可羧化的半抗原可使用混合酸干法與碳二亞胺法,戊二醛法與重氮化法用于含有氨基或者可還原硝基半抗原的小分子偶聯(lián),而含有羥基的半抗原可采用琥珀酸酐和羰基二咪唑作為偶聯(lián)臂。針對DBP小分子環(huán)境污染物,該研究選用BSA與OVA作為偶聯(lián)載體。在4-硝基鄰苯二甲酸的基礎(chǔ)上,首先創(chuàng)新型的選擇催化劑進(jìn)行酯化合成4-硝基鄰苯二甲酸二丁酯,然后選擇最優(yōu)的還原劑還原得到具有氨基結(jié)構(gòu)的4-氨基鄰苯二甲酸二丁酯,最后通過重氮化反應(yīng)偶聯(lián)蛋白載體獲得2種人工完全抗原。DBP-BSA作為免疫小鼠的免疫抗原,DBP-OVA作為建立方法的包被抗原。優(yōu)化實(shí)驗(yàn)條件后,該合成方法較文獻(xiàn)中的方法產(chǎn)率提高,合成時間縮短。AFTM1-BSA和AFTM1-OVA的2種小分子環(huán)境污染物人工完全抗原則通過購買獲得,前者作為免疫抗原,后者作為包被抗原。二.兩種小分子環(huán)境污染物單克隆抗體的制備與檢測方法的建立來自于同一株細(xì)胞的單克隆抗體比來自于不同只動物獲得的多克隆抗體相比,具有更高的特異性、更好的均一性并且可以體內(nèi)外大量制備,其來源和產(chǎn)量豐富等優(yōu)點(diǎn)。因此我們采用經(jīng)典的細(xì)胞融合的方法篩選得到陽性雜交瘤細(xì)胞并通過體內(nèi)培養(yǎng)、體外純化獲得足量的單克隆抗體。繼而通過優(yōu)化包被抗原濃度、抗體濃度與抗體稀釋液等多個條件建立最優(yōu)化的間接競爭ELISA (indirect complete enzyme-linked immunosorbent assay, Ic-ELISA)檢測方法。在競爭ELISA的方法中,采用制備得到的DBP-BSA抗原免疫BALB/c小鼠,經(jīng)細(xì)胞融合后經(jīng)4次篩選得到雜交瘤陽性細(xì)胞株,然后體內(nèi)大量制備單克隆抗體。在0.25 ug/ml的抗原包被濃度下,采用0.1%明膠稀釋液稀釋抗體16000倍,該方法靈敏度最高：IC50= 7.34 ng/ml,線性范圍為0-50 ng/ml, R2=0.994,最低檢測限為0.06 ng/ml,變異系數(shù)范圍為5.93-11.09%,回收率范圍97.8-114.3%。同時與GC-MS傳統(tǒng)儀器分析法相關(guān)性良好,且較文獻(xiàn)中其它各種方法相比,具有更強(qiáng)的特異性、更高的靈敏度、更好的精密度和準(zhǔn)確度。依托篩選得到的陽性雜交瘤細(xì)胞制備AFTM1單克隆抗體,優(yōu)化ELISA實(shí)驗(yàn)后最佳包被抗原濃度為0.5 ug/ml,抗體稀釋2000倍,方法的最低檢測限為4 ng/L,IC50為0.36 ng/ml,可接受變異系數(shù)和回收率分別為1.788-8.678%,94.21-102.3%。該方法比其他文獻(xiàn)檢測方法相比,具有靈敏度高、特異性強(qiáng)等優(yōu)點(diǎn)。根據(jù)兩種小分子環(huán)境污染物的的單克隆抗體建立的ELISA方法除了特異性好、靈敏度高、操作簡單以外,與儀器分析方法相比極大的提高了檢測的通量,可用于大量樣本中環(huán)境污染物質(zhì)的檢測。三.兩種小分子環(huán)境污染物人體內(nèi)暴露的檢測與分析根據(jù)上述建立的方法,對1246個普通人群樣本進(jìn)行DBP和AFTM1內(nèi)暴露含量的檢測,檢測數(shù)據(jù)采用PRISM和SPSS軟件進(jìn)行統(tǒng)計分析。DBP在人體尿樣樣本中的檢出率為72.87%,所有檢測樣本的幾何平均值為3 ng/ml。女性、中年組和低教育程度的人群分別較男性、老人組和高教育組人群含量高。AFTM1的檢測范圍為0.0004094-0.9284 ng/ml,檢出率和幾何平均值分別為83.01%與0.09229 ng/ml.通過性別、文化程度與年齡分析,結(jié)果顯示男性、中年組和低教育人群含量較高。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R114
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R114
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 王雯;李崗;魏云瀟;;我國食品中黃曲霉毒素污染現(xiàn)狀的研究[J];安徽農(nóng)業(yè)科學(xué);2015年18期
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