【作者】 陈寒玉；

【导师】 庄惠生；

【作者基本信息】 东华大学 ， 环境科学与工程， 2009， 博士

【摘要】 环境激素是一类会使人类及生物的内分泌系统发生紊乱的外来物质,它可以影响人类和生物的内分泌系统,导致生殖、发育和行为异常。多氯联苯类化学物质曾被作为优质工业添加剂,大量使用在石油产品、塑料、农药加工等行业。但毒理学研究表明,此类物质具有环境激素的特征,具有一定的致癌性和影响动物体的生殖和发育系统。当今存在的多氯联苯污染物,一部分是由于上世纪遗留下来的,另一部分是由新的污染源产生。由于多氯联苯分子结构的稳定性,使它不易被自然降解和消除,因此,如今在世界的各个地方几乎都能找到它的踪迹,成为一类普遍的环境污染物。多个国家都把它列为优先检测污染物并作为环境监测必检项目,并以严格的排放标准限制它的污染源排放。当前,环境中多氯联苯的检测方法主要有气相色谱、液相色谱和气质联用等色谱分析技术。但色谱技术需要复杂的前处理技术和昂贵的仪器,对操作人员的技术要求也比较高,因此不适于大规模环境样品的现场监测和批量监测。免疫分析技术具有方便快捷、检测成本低、灵敏度高等优点,可以满足简单、快速、灵敏检测环境激素的要求。目前已经有以ELISA为主的多种多氯联苯免疫监测方法,如分子传感器,表面细胞质团共振检测(SPR)等。免疫PCR是1992年由Sano首次提出的,它将体系完善的ELISA分析方法和具有强大信号放大能力的PCR技术结合起来。大量的应用研究表明,该方法与ELISA相比不仅具有很好的一致性而且灵敏度更高,线性范围更广,具有更强的检测能力。免疫PCR在技术和信号检测仪器上的进一步发展,导致了实时定量免疫PCR技术(rt-IPCR)的产生。实时荧光定量免疫PCR是近年来在荧光PCR和免疫PCR基础上建立起来的一种免疫检测新技术。此方法建立以来,已经在癌症、自身免疫缺陷、致病菌和细菌毒性等生物学和医学研究领域得到广泛应用,但是还没有见到有应用在多氯联苯污染物检测方面的报道。该技术和一般免疫PCR与传统的ELISA相比,具有更小的实验室内部误差和更高的灵敏度。因此本论文意在把该技术引入环境学研究领域,建立一种灵敏度更好的检测多氯联苯类环境污染物的新方法,为痕量有机污染物的检测提供一个新思路,对环境中多氯联苯的迁移、转化和降解等环境行为的调查研究提供一种新的检测方法。本文以多氯联苯类环境激素作为检测对象,选择出三种有代表性的多氯联苯单体,根据免疫检测的技术要求,主要通过半抗原衍生物的设计和制备、全抗原和抗体的制备与表征,采用荧光免疫PCR技术,分别建立了三种多氯联苯类环境激素的免疫检测新方法,并应用于实际样品的测定。课题的主要研究内容为：1.多氯联苯单体的合成方法研究：通过联苯胺还原法和改性Gomberg不对称合成法,制备出四种多氯联苯单体,产物通过红外色谱和元素分析进行了表征。通过对三种多氯联苯单体的合成,确立了一种Gomberg不对称合成多氯联苯新方法。2.多氯联苯单体半抗原的合成：通过Friedel-Crafts酰基化反应,在三种多氯联苯分子上引入羧基,合成了PCB12, PCB37, PCB77三种多氯联苯的半抗原衍生物。半抗原的分子结构和化学组成通过了~1H-NMR、IR和元素分析进行表征和确证。3.免疫原的制备：用活化酯法分别将三种半抗原与牛血清蛋白(BSA)偶联,制得PCBs的免疫原；用混合酸酐法将半抗原分别与卵清蛋白(OVA)偶联,制得PCBs的包被原。产物经紫外光谱、蛋白质含量测定等方法给予确证。4.多克隆抗体的制备：通过免疫新西兰大白兔,对血清进行分离和提纯,制备了三种效价高、特异性好的抗多氯联苯类环境激素的新抗体IgG；琼脂双向扩散实验表明PCB12,37,77的抗体效价分别为：1：32,1：32,1：64。5.生物素化探针DNA的制备和纯化：探针DNA是以pUC19质粒为模板扩增得到的一段103个碱基对的生物素化DNA。根据DNA扩增试剂盒的说明,在PCR扩增体系中加入各种反应物,包括两条生物素化了的引物。引物序列分别是：上游引物序列从5’到3’是G TAA AAC GAC GGC CAG T,下游引物的序列从5’到3’是CAG GAA ACA GCT ATG AC。PCR扩增的程序是：940C预变性4 min。然后是30个循环反应,每个循环的程序是94℃变性20s,55℃退火20s,72℃延伸20s。30个循环反应后在72℃保持3 min,使延伸完全。使用UNIQ-10 PCR纯化试剂盒,对PCR扩增产物进行纯化后,再用琼脂糖凝胶电泳后染色进行定性,并用紫外分光光度法定量检测分析。6.生物素化抗体和半抗原的制备：通过活化生物素法,使三种抗体与活化生物素(BNHS)进行偶联,产物经过透析纯化后,得到三种PCBs的特异性生物素化抗体；把活化生物素(BNHS)通过混合酸酐法与三种多氯联苯半抗原衍生物进行合成反应,得到了三种生物素化半抗原。产物经紫外光谱、蛋白质含量测定等方法给予确证；7.建立了间接竞争实时定量免疫聚合酶链式反应(间接竞争rt-IPCR)分析方法,用于检测环境中三种PCBs。用制备的包被原包被经过戊二醛处理的PCR小管,接着用PCBs与包被原竞争结合特异性抗体,再利用生物素化二抗与结合在包被原上的特异性抗体结合,然后通过亲和素与制备的生物素化的103bp探针DNA结合,最后通过实时定量PCR仪在优化的扩增程序下扩增DNA并实时检测。此方法对PCB12, PCB37, PCB77测定的标准曲线相关系数分别为：0.941,0.961,0.954。对三种多氯联苯的检测限分别达到1.75、1.32和1.50 fg／mL。8.建立了直接竞争实时定量免疫聚合酶链式反应(直接竞争rt-IPCR)分析方法,用于检测环境中三种PCBs。用制备的包被原包被经过戊二醛处理的PCR小管,接着用PCBs与包被原直接竞争结合生物素化特异性抗体,然后通过亲和素与制备的生物素化的DNA探针结合,最后通过实时定量PCR仪在优化的扩增程序下扩增DNA并实时检测。此方法对PCB12, PCB37, PCB77测定的标准曲线相关系数分别为：0.987,0.988,0.985。对三种多氯联苯的检测限分别达到1.53、1.42和1.25 fg／mL。9.建立了抗体包被实时定量免疫聚合酶链式反应(抗体包被rt-IPCR)分析方法,用于检测环境中三种PCBs。用制备的特异性抗体包被经过戊二醛处理的PCR小管,接着用PCBs与生物素化半抗原竞争结合特异性抗体,然后通过亲和素与制备的生物素化的DNA探针结合,最后通过实时定量PCR仪在优化的扩增程序下扩增DNA并实时检测。此方法对PCB12, PCB37, PCB77测定的标准曲线相关系数分别为：0.966,0.971,0.985。对三种多氯联苯的检测限分别达到1.75、1.45和1.33fg/mL。比较这三种方法可知,间接竞争rt-IPCR的检测灵敏度和工作曲线的相关性略低于其它两种方法。直接竞争rt-IPCR和抗体包被rt-IPCR均有较好的工作曲线相关性。通过对检测过程各步的优化发现,当采用0.8%的戊二醛溶液处理聚丙烯PCR管后,可以提高对抗原抗体的吸附能力,同时各种包被介质和包被时间、最佳抗原抗体结合浓度,包被液和封闭液、以及亲和素和生物素化DNA探针的浓度被优化,这些优化措施提高了检测的准确性。方法的特异性、回收率也被加以研究,并应用于实际土壤和污泥等环境样品的检测。结果表明,各种方法的交叉反应率小于15%,回收率在85～110%之间,样品的检测结果与GC／MS法的测定结果相比,具有良好的一致性。本论文建立的三种检测多氯联苯rt-IPCR方法均未见文献报道。更多还原

【Abstract】 "Environmental hormones" is a term given to endocrine disruptors, which are chemical substances capable of disordering animal hormones. Hence, it adversely affects the endocrine system and consequently alters the development and/or reproduction of organisms, including humans. Polychlorinated biphenyls (PCBs) are routinely used as additives in various oil-based preparations and are important in the manufacture of products as diverse as plastics and pesticides. The toxicological study indicates that the PCBs mimic, impair, enhance or inhibit the actions of endogenous hormones, which cause malignant tumors and abnormalities in growth and reproduction.Today, the PCBs presenting in the environment mainly root in the leftover in last century, and still partly sourced from nature and anthropogenic processes. Due to the stability of chemical structure, the PCBs are uneasily removed by the sunlight and microbe. Therefore the PCBs distribute worldwide in the environment and become one of the most prevalent pollutants. It has been banned in industrialized countries and monitored routinely as priority pollutant for regulatory purposes, and the stringent discharge standards are issued to control the new PCBs in the environment.Gas chromatography, high performance liquid chromatography and high-resolution gas chromatography/mass spectrometry (HGGC/HGMS) are the usual methods for determination of PCBs. However there are some shortcomings in these methods, such as expensive instruments, professional techniques, infeasibility of the on-site analyses and batch testing in practice samples. In contrast, immunoassays are typically very sensitive and readily adapted to analysis for which an appropriate antibody is available. Nowadays, there were many reliable immune methods in detecting PCBs, such as ELISAs, biosensor and SPR. In this paper, we will use a new immunoassay for detecting PCBs in the environment.The IPCR method, first described by Sano et al. in 1992, combines the well-established ELISA methodology with the signal amplification power of the PCR. A number of research applications describe the advantages of the method, that is, in particular, its high sensitivity and good quantification capabilities due to the great linearity and compatibility with established ELISA protocols. Further development in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of real-time IPCR (rt-IPCR).Real-time immuno-polymerase chain reaction (rt-IPCR) is a versatile and robust technique based on the advantage of rt-PCR and IPCR for the ultra-sensitive analysis of pathological proteins and other viral antigens. With its development, the method has been applied to detect antigens associated with cancer, autoimmune diseases, pathogenic bacteria and bacterial toxin in clinical diagnosis and biochemical analysis fields. But no environmental pollutants have been detected by rt-IPCR. The method’s sensitivity was higher over conventional ELISA according to the reports, and inter-assay error was lower than IPCR. This paper was to introduce this versatile technique into environmental monitoring field and set up a series of new immunoassays for determination of trace environmental hormone.In this work, three kinds of representative PCBs congeners were selected and modified for the immunoassay study. We designed and prepared optimum haptens acting as immunogens and competitors, and then characterized the artificial antigen and antibody. A series of rt-IPCR immunoassays were developed to detect PCBs in real environmental samples. The work is summarized as the following:1. Study on the methods of synthesis PCBs congeners. One PCB15 derivatives was synthesized by the method of deoxidized biphenyl amine, and three congeners were synthesized by the modified Gomberg-Bachmann reaction. All PCB congeners were characterized by IR, ~1HNMR and element analysis. A new synthesis PCB congener method was developed.2. Three PCB congener haptens were synthesized by the Friedel-crafts acylation reaction and a carboxyl was inducted to the PCB( PCB12,PCB37,PCB77) molecule. And the products were characterized by IR, ~1HNMR and element analysis technology.3. Synthesis of artificial antigens. The haptens were conjugated to the carrier proteins (BSA, OVA) with the modified active ester method or with the mixed acid anhydride method to form immune antigens and coating antigens. In this way the artificial antigen of PCBs were obtained. Structures of the products were characterized by UV spectra and detecting protein contentration.4. Preparation of polyclonal antibodies. Male New Zealand white rabbits were immunized with the mixture of immune antigens and freund adjuvant. The antiserum from the rabbit was purified by the method of octanoic acid ammonium sulfate two-step precipitation, Sephadex G-25 and DEAE cellulose. In this way the specific and affinitive pAbs of PCBs had been yielded. The sera were tested for titer by the agar diffusion test and the tube agglutination reaction test, the titer of the antibody for the PCB12,37,77 is 1:32,1:32,1:64 respectively.5. Preparation and purification of biotinylated reporter DNA:The reporter DNA with biotin is a 103-base pair sequence from the pUC19 vectors. The reporter DNA was generated by PCR as follows. The forward primer, M13/pUC sequencing as G TAA AAC GAC GGC CAG T, was biotinylated at the 5’ end to generate a biotiny group to the DNA. The reverse primer, M13/pUC reverse sequencing as CAG GAA ACA GCT ATG AC was also biotinylated at the 5’ end to generate a biotiny group to the DNA. All regents were added to PCR tube as described by the DNA PCR kit handbook. The PCR conditions were: hold 94℃for 4 min; 30 cycles of 94℃for 20 s, 55℃for 20 s, and 72℃for 20 s. The 72℃step is extended to 3 min in the final cycle. The reporter DNA was purified and retrieved by UNIQ-10 PCR DNA extraction kit. The DNA was quantified by UV absorbency and checked qualitatively using agarose gel.6. Preparation of biotinylated polyclonal antibodies and haptens. The specific and affinitive pAbs yielded in our own laboratory were biotinylated, using biotinamido-caproate-N-hydroxysuccinimide ester (BNHS) as reported. The biotinylated antibody was then purified by the semi permeable membrane dialyzing. The biotinylated haptens were synthesized by the mixed acid anhydride method using the three PCB haptens with BNHS. Structures of the products were characterized by UV spectra and detecting protein content ration.7. Indirect competitive real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde, was used to compete with the PCBs in combining pAbs. Biotinylated goat anti-rabbit IgG were added to combine pAbs. Avidin was used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated 103 bp reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.978, 0.961, 0.954 were obtained according to the PCB12, PCB 37, PCB 77.8. Direct competitive real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde, was used to competing with the PCBs in combining biotinylated pAbs. Avidin was used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.987, 0.988, 0.985were obtained according to the PCB12, PCB 37, PCB 77.9. Antibody-coated real-time Immuno-PCR (rt-IPCR) assay for the determination of PCBs in environment was developed. Glutaraldehyde treated PCR tubes were adsorbed by coating pAbs specific for PCBs, with which biotinylated PCBs hapten and antigen (Ag) PCBs were competing in combining. Avidin was used as a bridge between the biotinylated PCBs hapten and the biotinylated reporter DNA. The reporter DNA was amplified and measured by rt-PCR under the optimize procedure. The correlative coefficients of 0.966, 0.971 , 0.985 were obtained according to the PCB12, PCB 37, PCB 77.Comparing the results of the three methods, we will find that the direct competitive rt-IPCR and antibody-coated rt-IPCR has better performance than the indirect competitive rt-IPCR. The reagent preparation and the assay characterization. optimization and validation steps are described. The optimum experimental situation is systematically studied with coating medium and time, the concentration of antibody, biotinylated DNA and haptens, et al respectively. After PCR tubes treated with 0.8% glutaraldehyde, the increasing absorb capability of tubes can improve the veracity of the assay. The coating medium and time are investigated, the results show that the pH 9.6 CBS is optimal coating solvent, the coating time is suitably in 4°C throughout the night, and the 3% OVA was the optimal blocking solvent. Cross-reaCtivity, recovery rates and detection limit are also observed. The results show that the cross-reactivity of the rt-IPCR is less than 15%. Good recoveries (85 - 110%) are obtained for spiked purified soil extracts. The analytical performance of the assay is consistent with GC/MS. These data indicate that the rt-IPCR is suitable for screening PCBs in environment, and there is no reports about these methods that have been detected.更多还原