环境污染物质检测技术研究及几种环境污染物质的内暴露分析
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摘要
环境污染问题是全世界所面临的重大问题之一。一般而言,环境污染的趋势随着经济的发展而加深,特别是工业革命期间,“先污染、后治理”的理念使得环境质量急剧下降。近年来,虽然人们的环境保护意识在不断提高,但是在经济利益的驱使下,每天仍有各种有害物质在超负荷排放并且更有大量未知的新化合物在大量涌现,这些环境污染物会对人类的健康生活造成具大的威胁。据中国2012年发布的((2012中国肿瘤登记年报》中的数据显示,我国每年新发肿瘤病例约为312万例,平均每天8550人,每分钟就有6人确诊为癌症。全国肿瘤死亡率为180.54/10万,每年因癌症死亡病例达270万例。癌症已经成为一个主要的公共健康问题。
虽然导致疾病和癌症的因素多种多样,但环境污染是不可忽视的重要的致病因素之一。目前我国对环境污染物的监测主要是针对外环境,这些监测的数据可以间接反应污染物质的暴露来源,为控制人和动物的过多暴露提供间接依据。而直接对人体内污染物质的含量进行检测能避免个体差异的影响,不仅能直接反映个体的污染物内暴露水平,为污染物的预警和减排处置提供直接依据,而且能为制订环境污染物质人体接触限值标准提供依据。但是,由于环境污染物种类多,人类暴露存在不确定性。利用常规仪器分析方法无疑是重成本的大海捞针,检测通量低以及灵敏度问题极大的限制了环境污染物质内暴露检测的发展。而各种突发的污染事件以及内暴露的未知性使得人们对环境污染的意识忽而过度敏感忽而麻木不仁,而且目前关于污染物质人体接触限值标准也没有明确规定。这些问题都是提高全民生活质量和健康水平的一大障碍。因此,为了解决环境污染物质内暴露检测的瓶颈问题,在本研究中,我们首先从提高检测通量以及灵敏度两方面入手,建立了多种环境污染物质的内暴露检测方法。其次,将建立的方法应用于人体样本,检测了多种环境污染物质在人体内的暴露水平,初步反映了中国人群在环境污染物质中的暴露程度及分布特征,建立了相应的基础数据库。最后,我们还以本研究中的人群为对象,初步对几种环境污染物质与人类健康状况进行了相关性分析。
1.几种常见环境污染物质多克隆抗体的制备及ELISA检测方法的建立
背景：目前针对环境污染物质的检测方法主要有气相色谱法(GC)、液相色谱法(LC)、高效液相色谱法(HPLC)和高效液相色谱-质谱联用法(LC-MS/MS),-方面这些仪器分析方法前处理复杂、耗时多,成本高且通量低,不适合大量样本的检测,另一方面对环境污染物质的检测主要是针对外环境的检测而很少涉及内暴露检测。目的：本研究的目的在于建立一些基于抗原抗体特异性结合原理的低成本、高通量的ELISA检测方法用于人体尿样中环境污染物质的检测。这几类环境污染物质包括：柠檬黄(TAR)、可替宁(COT)、双酚A (BPA)、铅(Pb)、镉(Cd)和铜(Cu)。方法：对于柠檬黄、可替宁和双酚A这三种物质,我们利用合成的完全人工抗原免疫新西兰大白兔,成功制备这三种物质的特异性多克隆抗体,并建立了相应的ELISA检测方法。针对铅、镉和铜等重金属污染物,我们购买了商品化的单克隆抗体,基于这些抗体建立了3种人体尿样中重金属含量的间接竞争ELISA检测方法。结果：柠檬黄间接竞争ELISA中,柠檬黄抗体ICs0为7.7ng/ml,柠檬黄的最低检测限为0.04ng/ml,方法回收率在99.8%到106.6%之间,变异系数在3.28%到14.9%之间,未见其与七种其他人工染料出现交叉反应。针对可替宁多克隆抗体,我们同时建立了间接竞争ELISA和直接竞争ELISA方法,在COT间接竞争ELISA中,抗体的ICso为15ng/ml, COT的最低检测限为0.1ng/ml.在COT直接竞争ELISA中,抗体的ICs0为7.1ng/ml, COT的最低检测限为0.04ng/ml,回收率在97.23%到111.26%之间,变异系数在4.63%到12.9%之间。针对双酚A多克隆抗体,我们同时建立了间接竞争ELISA和直接竞争ELISA方法,在BPA间接竞争ELISA中,抗体的IC50为7.0ng/ml, BPA的最低检测限为0.08ng/ml,回收率在100.99%-113.81%之间,变异异数在6.23%-14.58%之间。在BPA直接竞争ELISA中,抗体的IC50为5.5ng/ml, BPA的最低检测限为0.03ng/ml,97.34%-119.42%之间,变异异数在7.37%-15.46%之间。针对重金属间接竞争ELISA,铅的IC50和最低检测限分别为5.09ng/ml和0.01ng/ml,方法回收率在98.7%到108.3%之间,变异系数在2.83%到13.92%之间。镉的的IC50和最低检测限分别为10.1ng/ml和0.07ng/ml,方法回收率在95.6%到103.9%之间,变异系数在2.56%到12.99%之间。铜的IC50和最低检测限分别为7.3ng/ml和0.04ng/ml,方法回收率在93.2%到106.5%之间,变异系数在2.67%到14.23%之间。结论：结果显示,本研究建立的所有方法特异性好、灵敏度高、操作简单,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。
2.沙丁胺醇免疫PCR检测方法的建立
背景：随着人们对健康要求的不断提高,有必要建立一些更高灵敏度的检测方法用于环境污染物质的检测。目的：本研究的目的是建立一种新型的基于DNA标记的超灵敏免疫PCR方法检测低分子量环境污染物质的含量。方法：本研究以沙丁胺醇(SAL)为研究对象,针对传统免疫PCR进行改造,将待测小分子物质SAL与DNA片段进行偶联,首创性的建立了基于DNA免疫探针的直接免疫PCR法并首次用于SAL的检测,只需三步即可完成检测：即SAL抗体的包被——SAL与DNA标记SAL的竞争反应——PCR扩增。结果：在最佳条件下,方法的最低检测限为21fg/ml,线性范围达到7个数量级。此方法结合了免疫学的高特异性及PCR的高灵敏性,SAL在人尿样中的回收率在85.5%和98.3%之间,变异系数在5.76%和9.23%之间。结论：本研究制备的SAL免疫PCR检测结合了免疫反应的高特异性以及PCR反应的高灵敏度,大大提高了SAL的检测灵敏度,是目前所建立的SAL检测方法中灵敏度最高的方法,此方法经改造后能用于其它小分子化合物的检测,能领引小分子量化合物超高灵敏检测的趋势。
3.几种常见环境污染物质人体内暴露水平测定及其与健康状况的相关性初步研究
目的：了解几种常见环境污染物质(柠檬黄、可替宁、沙丁胺醇、铅和黄曲霉毒素M1)在人体内的含量,初步分析几种环境污染物质与检测人群健康状况的相关性。方法：用本研究中建立的ELISA检测方法检测柠檬黄、可替宁、沙丁胺醇、铅和黄曲霉毒素M1在研究人群尿样中的含量,得到结果后,采用SPSS软件对人群中污染物质的含量进行描述性及相关性分析。结果：柠檬黄在351例人体样本中的检测范围为0-109.4ng/ml;可替宁在951例人样本中的检测范围为0-6257ng/ml,几何平均值(GM)为10.32ng/ml;沙丁胺醇在951例人样本中的检测范围为0-16.06ng/ml, GM为0.61ng/ml;铅在951例人样本中的检测范围为0-139ng/ml, GM为0.23ng/ml。在沙丁胺醇、铅与健康状况的相关性分析中,我们没有发现明显相关性。结论：这几种环境污染物质在人体内均有不同程度的检出,环境污染问题应受到极大的重视,环境污染物质对人体的影响及其限值研究亟待加强。
综上所述：
本研究针对限制环境污染物质内暴露检测项目的两大问题进行了技术分析与攻关。对于检测通量的提高,我们以免疫学原理为基础,建立了6种不同环境污染物质的ELISA检测方法用于内暴露检测。这些方法不仅灵敏度高,特异性好,更重要的是在通量方面有了明显提升。对于灵敏度的提高,我们利用免疫学原理中的高特异性与PCR反应的高灵敏性,建立了针对于小分子量化合物的改良免疫PCR检测技术,该技术首次应用于小分子量化合物SAL的检测,在灵敏度方面有了极大的提升,是目前SAL检测方法中灵敏度最高的一种。此外,我们还初步检测了几种环境污染物质在人群中的暴露水平并分析了环境污染物质与人群健康状况的相关性。本研究首次系统的从技术研究到人群检测再到健康状况相关性分析三方面展开了对环境污染物质的内暴露研究,为环境污染物质内暴露检测项目在普通人群中的推广提供了技术支持和理论依据,调查了环境污染物质人体内暴露的人群分布特征,建立了相应的基础数据,为环境污染物质的人体内暴露限值标准的制订及其对疾病的相关性研究奠定基础。
Environmental pollution is one of the biggest problems worldwide. Generally speaking, the extent of environmental pollution is aggravated with the development of economy and industrialization. Especially, during the industrial revolution, the idea of "pollution first, treatment later" made a dramatic fall in the environmental quality. In recent years, although the consciousness of environmental protection is improving, but there are also different kinds of unknown new chemicals and hazardous substances that are excreted into the environment every day from different sources. These hazardous emissions pose a variety of potential threats to human health. According to the "2012Annual Report of Cancer Registration in China", China has about3.12million new cancer cases per year, an average of8550people per day,6people are diagnosed with cancer every minute. National cancer mortality was180.54/10million, which show that the annual deaths due to cancer reached2.7million peoples. Cancer has become a major public health problem.
Although there are various factors that can cause cancer, environmental pollution is one of the most important pathogenic factors that cannot be ignored. Nowadays, the monitoring of environmental pollutants is mainly focused on the external environment, which can indirectly reflect the sources of pollutants and provide basis for controlling the exposure level of environmental pollutants in human and animals. Detecting the concentration of environmental pollutants in human can avoid the influence of individual differences, directly reflect the exposure level of individual to pollutants. The data not only provide direct basis for early warning of pollutants and disposal but also provide the basis for the standard of human exposure limit. However, there are various types of environmental pollutants in environment and exposure of human to individual pollutant is undefined, so it's very difficult to detect all pollutants in human using conventional detection methods. The problem of low through-put and sensitivity is placing a big limit on the development of research about human exposure to environmental pollutants. All kinds of environmental pollution emergencies and unascertainty in human exposure to pollutants make people either supersensitive or insensible to environmental pollutants. It's a great barrier for developing high-quality health level for all people. Therefore, we dedicated to solve the bottleneck problem in the detection of human exposure to environmental pollutants. First of all, we established several methods to detect different environment pollutants, focusing on the high sensitivity and high throughput. Secondly, we used these methods to assess the exposure of human to these different environmental pollutants. The data reflect the exposure of the Chinese population to environmental pollutants and the distribution of exposure. This work can be used as a primary database about Chinese exposure to the environment pollutants. Finally, we also investigate the relationship about the pollutants exposure with human health status.
1. Preparation of antibodies against different environmental pollutants and development of enzyme-linked immuno sorbent assay (ELISA) for detection of these pollutants
Background:Various analytical techniques and methods have been developed for the detection of environmental pollutants including gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC) and HPLC combined with tandem mass spectrometry (LC-MS/MS). On one hand, these instrumental analyses require complex pretreatment, high cost and relatively time-consuming; on the other hand, most methods are focused on the detection of pollutants in external environment.
Objectives:Therefore, we aimed to develop some low cost and high throughput methods based on specific immunoreactions to assess the exposure of human to different environmental pollutants, including tartrazine (TAR), cotinine (COT), bisphenol A (BPA), lead (Pb), cadmium (Cd) and copper (Cu).
Methods:For TAR, COT and BPA, we synthesized artificial antigens and prepared three specific polyclonal antibodies using synthesized artificial antigens. For Pb, Cd and Cu, we purchased commercial monoclonal antibodies, and developed three indirect competitive ELIS As for detection of urinary concentration of Pb, Cd and Cu based on three monoclonal antibodies.
Results:For TAR, we developed an indirect competitive ELISA for detection of TAR in human urinary samples. The IC50value of TAR-antibody was7.7ng/ml; the limit of detection (LOD) was set at0.04ng/ml; the recoveries of TAR were from99.8%to106.6%and coefficients of variation were from3.28%to14.9%; no cross-reactivity of the antibody was observed with seven other artificial dyes based on this assay.
For COT, we developed and compared direct and indirect competitive ELISAs for detection of COT in human urine. The IC50value and LOD for indirect competitive ELISA were15ng/ml and0.1ng/ml respectively, whereas for the direct competitive, the IC50value and LOD were7.1ng/ml and0.04ng/ml respectively, the recoveries of COT were from97.23%to111.266%and coefficients of variation were from4.63%to12.9%.
For BPA, we developed and compared direct and indirect competitive ELISAs for the detection of BPA in human urine. The IC50value and LOD for the indirect competitive ELISA were7.0ng/ml and0.08ng/ml respectively, the recoveries of BPA were from100.99%to113.81%and coefficients of variation were from6.23%to14.58%, whereas for the direct competitive, the The IC50value and LOD for the indirect competitive ELISA were5.5ng/ml and0.03ng/ml respectively; the recoveries of BPA were from97.34%to119.42%and coefficients of variation were from7.37%to 15.46%.
The IC50value and LOD of Pb for indirect competitive ELISA were5.09ng/ml and0.01ng/ml respectively, the recoveries of Pb were from98.7%to108.3%and coefficients of variation were from2.83%to13.92%. The IC50value and LOD of Cd for indirect competitive ELISA were10.1ng/ml and0.07ng/ml respectively, the recoveries of Cd were from95.6%to103.9%and coefficients of variation were from2.56%to12.99%. The IC50value and LOD for the indirect competitive ELISA about Cu were7.3ng/ml and0.04ng/ml respectively, the recoveries of Cu were from93.2%to106.5%and coefficients of variation were from2.67%to14.23%.
Conclusions'. The results indicated that all the methods developed in this research were highly specific, highly sensitive and easy-to-use, and could be used for the detection of environmental pollutants in a large number of human samples.
2. Development of Immuno-PCR assay for detection of SAL
Background:It's necessary to develop a more sensitive method to detect the environmental pollutants with the constant improvement of human health needs.
Objectives:The aim of our study was to develop a novel pre-assembled DNA immunoprobe based direct competitive immuno-PCR for the determination of SAL level in human urine.
Methods:In this study, we focused on SAL, modified the traditional immuno-PCR, conjugated the analyte SAL to reporter DNA and for the first time we have developed a novel pre-assembled DNA immunoprobe based direct competitive immuno-PCR to investigate the level of small molecule SAL in human urine. There were only three steps in this assay, namely SAL antibody coating-competition between free SAL and DNA conjugated SAL-PCR amplification.
Results:Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, and the limit of detection of SAL in human urine was found to be21fg/ml, the recoveries of SAL were from85.5%to98.3%and coefficients of variation were from5.76%to9.23%.
Conclusions:This method combines the specificity of immunoassays with PCR signal amplification and provides a huge improvement of sensitivity of SAL. To the best of our knowledge, the sensitivity in this study is one of the highest sensitivity observed for detecting SAL. Moreover, this method can be modified and used for detecting other small molecule chemicals in environment and could lead the trend to the ultrasensitive detection of small molecules chemicals.
3. Determination of internal exposure of human to different environmental pollutants and preliminary correlation analysis between environmental pollutants and human disease
Objective:To understand the exposure to several common environmental pollutants in general population and preliminary study correlation between environmental pollutants and human disease.
Methods:Using the methods developed in this research, we measured the urinary concentrations of TAR, COT, SAL, Pb and AFM1in the selected participants. After detection, descriptive and correlation analysis were conducted by SPSS.
Results:The detection range for TAR in351participants was0-109.4ng/ml; the GM for COT in951participants was10.32ng/ml, the detection range was0-6257ng/ml; the GM for SAL in951participants was0.61ng/ml, the detection range was0-16.06ng/ml; the GM for Pb in951participants was0.23ng/ml, the detection range was0-139ng/ml. In the correlation analysis between environmental pollutants and human disease, we found higher urinary COT concentrations were associated with lung cancer.
Conclusions:All environmental pollutants in our research can be detected in the selected participants, and further studies are needed to reveal the effects of chronic exposure on human.
To sum up:
In our study, we explored two problems that limited the development of research about human exposure to environmental pollutants. To improve the detection throughput, we have established six different methods to detect human exposure to environmental pollutants based on ELISA. These methods have high sensitivity and specificity, more importantly, the throughput of detection has improved significantly. For the improvement of the sensitivity, we combined the high specificity of immunological and high sensitivity of the PCR reaction, and developed the modified immuno-PCR which is used specific to the detection of low molecular weight compound. The modified immuno-pcr was first applied to the detection of a low molecular weight compound (SAL) in our study. The method shows a super highly improvement to sensitivity and is found to be the most sensitive method to SAL detection. In addition, we also preliminary detected human exposure to several different environmental pollutants and analyzed the relationship between environmental pollutants and population health. This is a first systematic study about human exposure to environmental pollutants which from technical research to detection on human exposure to correlation analysis between the health and human exposure to pollutants. This study provided technical supports and theoretical basis for the generalization of detection about internal exposure, investigated the population distribution to environmental pollutants, established the corresponding primary database of human exposure level to environmental pollutants, and laid the foundation to the fomulation of limit value standard and correlation analysis between the human diseases and environmental pollutants.
虽然导致疾病和癌症的因素多种多样,但环境污染是不可忽视的重要的致病因素之一。目前我国对环境污染物的监测主要是针对外环境,这些监测的数据可以间接反应污染物质的暴露来源,为控制人和动物的过多暴露提供间接依据。而直接对人体内污染物质的含量进行检测能避免个体差异的影响,不仅能直接反映个体的污染物内暴露水平,为污染物的预警和减排处置提供直接依据,而且能为制订环境污染物质人体接触限值标准提供依据。但是,由于环境污染物种类多,人类暴露存在不确定性。利用常规仪器分析方法无疑是重成本的大海捞针,检测通量低以及灵敏度问题极大的限制了环境污染物质内暴露检测的发展。而各种突发的污染事件以及内暴露的未知性使得人们对环境污染的意识忽而过度敏感忽而麻木不仁,而且目前关于污染物质人体接触限值标准也没有明确规定。这些问题都是提高全民生活质量和健康水平的一大障碍。因此,为了解决环境污染物质内暴露检测的瓶颈问题,在本研究中,我们首先从提高检测通量以及灵敏度两方面入手,建立了多种环境污染物质的内暴露检测方法。其次,将建立的方法应用于人体样本,检测了多种环境污染物质在人体内的暴露水平,初步反映了中国人群在环境污染物质中的暴露程度及分布特征,建立了相应的基础数据库。最后,我们还以本研究中的人群为对象,初步对几种环境污染物质与人类健康状况进行了相关性分析。
1.几种常见环境污染物质多克隆抗体的制备及ELISA检测方法的建立
背景：目前针对环境污染物质的检测方法主要有气相色谱法(GC)、液相色谱法(LC)、高效液相色谱法(HPLC)和高效液相色谱-质谱联用法(LC-MS/MS),-方面这些仪器分析方法前处理复杂、耗时多,成本高且通量低,不适合大量样本的检测,另一方面对环境污染物质的检测主要是针对外环境的检测而很少涉及内暴露检测。目的：本研究的目的在于建立一些基于抗原抗体特异性结合原理的低成本、高通量的ELISA检测方法用于人体尿样中环境污染物质的检测。这几类环境污染物质包括：柠檬黄(TAR)、可替宁(COT)、双酚A (BPA)、铅(Pb)、镉(Cd)和铜(Cu)。方法：对于柠檬黄、可替宁和双酚A这三种物质,我们利用合成的完全人工抗原免疫新西兰大白兔,成功制备这三种物质的特异性多克隆抗体,并建立了相应的ELISA检测方法。针对铅、镉和铜等重金属污染物,我们购买了商品化的单克隆抗体,基于这些抗体建立了3种人体尿样中重金属含量的间接竞争ELISA检测方法。结果：柠檬黄间接竞争ELISA中,柠檬黄抗体ICs0为7.7ng/ml,柠檬黄的最低检测限为0.04ng/ml,方法回收率在99.8%到106.6%之间,变异系数在3.28%到14.9%之间,未见其与七种其他人工染料出现交叉反应。针对可替宁多克隆抗体,我们同时建立了间接竞争ELISA和直接竞争ELISA方法,在COT间接竞争ELISA中,抗体的ICso为15ng/ml, COT的最低检测限为0.1ng/ml.在COT直接竞争ELISA中,抗体的ICs0为7.1ng/ml, COT的最低检测限为0.04ng/ml,回收率在97.23%到111.26%之间,变异系数在4.63%到12.9%之间。针对双酚A多克隆抗体,我们同时建立了间接竞争ELISA和直接竞争ELISA方法,在BPA间接竞争ELISA中,抗体的IC50为7.0ng/ml, BPA的最低检测限为0.08ng/ml,回收率在100.99%-113.81%之间,变异异数在6.23%-14.58%之间。在BPA直接竞争ELISA中,抗体的IC50为5.5ng/ml, BPA的最低检测限为0.03ng/ml,97.34%-119.42%之间,变异异数在7.37%-15.46%之间。针对重金属间接竞争ELISA,铅的IC50和最低检测限分别为5.09ng/ml和0.01ng/ml,方法回收率在98.7%到108.3%之间,变异系数在2.83%到13.92%之间。镉的的IC50和最低检测限分别为10.1ng/ml和0.07ng/ml,方法回收率在95.6%到103.9%之间,变异系数在2.56%到12.99%之间。铜的IC50和最低检测限分别为7.3ng/ml和0.04ng/ml,方法回收率在93.2%到106.5%之间,变异系数在2.67%到14.23%之间。结论：结果显示,本研究建立的所有方法特异性好、灵敏度高、操作简单,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。
2.沙丁胺醇免疫PCR检测方法的建立
背景：随着人们对健康要求的不断提高,有必要建立一些更高灵敏度的检测方法用于环境污染物质的检测。目的：本研究的目的是建立一种新型的基于DNA标记的超灵敏免疫PCR方法检测低分子量环境污染物质的含量。方法：本研究以沙丁胺醇(SAL)为研究对象,针对传统免疫PCR进行改造,将待测小分子物质SAL与DNA片段进行偶联,首创性的建立了基于DNA免疫探针的直接免疫PCR法并首次用于SAL的检测,只需三步即可完成检测：即SAL抗体的包被——SAL与DNA标记SAL的竞争反应——PCR扩增。结果：在最佳条件下,方法的最低检测限为21fg/ml,线性范围达到7个数量级。此方法结合了免疫学的高特异性及PCR的高灵敏性,SAL在人尿样中的回收率在85.5%和98.3%之间,变异系数在5.76%和9.23%之间。结论：本研究制备的SAL免疫PCR检测结合了免疫反应的高特异性以及PCR反应的高灵敏度,大大提高了SAL的检测灵敏度,是目前所建立的SAL检测方法中灵敏度最高的方法,此方法经改造后能用于其它小分子化合物的检测,能领引小分子量化合物超高灵敏检测的趋势。
3.几种常见环境污染物质人体内暴露水平测定及其与健康状况的相关性初步研究
目的：了解几种常见环境污染物质(柠檬黄、可替宁、沙丁胺醇、铅和黄曲霉毒素M1)在人体内的含量,初步分析几种环境污染物质与检测人群健康状况的相关性。方法：用本研究中建立的ELISA检测方法检测柠檬黄、可替宁、沙丁胺醇、铅和黄曲霉毒素M1在研究人群尿样中的含量,得到结果后,采用SPSS软件对人群中污染物质的含量进行描述性及相关性分析。结果：柠檬黄在351例人体样本中的检测范围为0-109.4ng/ml;可替宁在951例人样本中的检测范围为0-6257ng/ml,几何平均值(GM)为10.32ng/ml;沙丁胺醇在951例人样本中的检测范围为0-16.06ng/ml, GM为0.61ng/ml;铅在951例人样本中的检测范围为0-139ng/ml, GM为0.23ng/ml。在沙丁胺醇、铅与健康状况的相关性分析中,我们没有发现明显相关性。结论：这几种环境污染物质在人体内均有不同程度的检出,环境污染问题应受到极大的重视,环境污染物质对人体的影响及其限值研究亟待加强。
综上所述：
本研究针对限制环境污染物质内暴露检测项目的两大问题进行了技术分析与攻关。对于检测通量的提高,我们以免疫学原理为基础,建立了6种不同环境污染物质的ELISA检测方法用于内暴露检测。这些方法不仅灵敏度高,特异性好,更重要的是在通量方面有了明显提升。对于灵敏度的提高,我们利用免疫学原理中的高特异性与PCR反应的高灵敏性,建立了针对于小分子量化合物的改良免疫PCR检测技术,该技术首次应用于小分子量化合物SAL的检测,在灵敏度方面有了极大的提升,是目前SAL检测方法中灵敏度最高的一种。此外,我们还初步检测了几种环境污染物质在人群中的暴露水平并分析了环境污染物质与人群健康状况的相关性。本研究首次系统的从技术研究到人群检测再到健康状况相关性分析三方面展开了对环境污染物质的内暴露研究,为环境污染物质内暴露检测项目在普通人群中的推广提供了技术支持和理论依据,调查了环境污染物质人体内暴露的人群分布特征,建立了相应的基础数据,为环境污染物质的人体内暴露限值标准的制订及其对疾病的相关性研究奠定基础。
Environmental pollution is one of the biggest problems worldwide. Generally speaking, the extent of environmental pollution is aggravated with the development of economy and industrialization. Especially, during the industrial revolution, the idea of "pollution first, treatment later" made a dramatic fall in the environmental quality. In recent years, although the consciousness of environmental protection is improving, but there are also different kinds of unknown new chemicals and hazardous substances that are excreted into the environment every day from different sources. These hazardous emissions pose a variety of potential threats to human health. According to the "2012Annual Report of Cancer Registration in China", China has about3.12million new cancer cases per year, an average of8550people per day,6people are diagnosed with cancer every minute. National cancer mortality was180.54/10million, which show that the annual deaths due to cancer reached2.7million peoples. Cancer has become a major public health problem.
Although there are various factors that can cause cancer, environmental pollution is one of the most important pathogenic factors that cannot be ignored. Nowadays, the monitoring of environmental pollutants is mainly focused on the external environment, which can indirectly reflect the sources of pollutants and provide basis for controlling the exposure level of environmental pollutants in human and animals. Detecting the concentration of environmental pollutants in human can avoid the influence of individual differences, directly reflect the exposure level of individual to pollutants. The data not only provide direct basis for early warning of pollutants and disposal but also provide the basis for the standard of human exposure limit. However, there are various types of environmental pollutants in environment and exposure of human to individual pollutant is undefined, so it's very difficult to detect all pollutants in human using conventional detection methods. The problem of low through-put and sensitivity is placing a big limit on the development of research about human exposure to environmental pollutants. All kinds of environmental pollution emergencies and unascertainty in human exposure to pollutants make people either supersensitive or insensible to environmental pollutants. It's a great barrier for developing high-quality health level for all people. Therefore, we dedicated to solve the bottleneck problem in the detection of human exposure to environmental pollutants. First of all, we established several methods to detect different environment pollutants, focusing on the high sensitivity and high throughput. Secondly, we used these methods to assess the exposure of human to these different environmental pollutants. The data reflect the exposure of the Chinese population to environmental pollutants and the distribution of exposure. This work can be used as a primary database about Chinese exposure to the environment pollutants. Finally, we also investigate the relationship about the pollutants exposure with human health status.
1. Preparation of antibodies against different environmental pollutants and development of enzyme-linked immuno sorbent assay (ELISA) for detection of these pollutants
Background:Various analytical techniques and methods have been developed for the detection of environmental pollutants including gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC) and HPLC combined with tandem mass spectrometry (LC-MS/MS). On one hand, these instrumental analyses require complex pretreatment, high cost and relatively time-consuming; on the other hand, most methods are focused on the detection of pollutants in external environment.
Objectives:Therefore, we aimed to develop some low cost and high throughput methods based on specific immunoreactions to assess the exposure of human to different environmental pollutants, including tartrazine (TAR), cotinine (COT), bisphenol A (BPA), lead (Pb), cadmium (Cd) and copper (Cu).
Methods:For TAR, COT and BPA, we synthesized artificial antigens and prepared three specific polyclonal antibodies using synthesized artificial antigens. For Pb, Cd and Cu, we purchased commercial monoclonal antibodies, and developed three indirect competitive ELIS As for detection of urinary concentration of Pb, Cd and Cu based on three monoclonal antibodies.
Results:For TAR, we developed an indirect competitive ELISA for detection of TAR in human urinary samples. The IC50value of TAR-antibody was7.7ng/ml; the limit of detection (LOD) was set at0.04ng/ml; the recoveries of TAR were from99.8%to106.6%and coefficients of variation were from3.28%to14.9%; no cross-reactivity of the antibody was observed with seven other artificial dyes based on this assay.
For COT, we developed and compared direct and indirect competitive ELISAs for detection of COT in human urine. The IC50value and LOD for indirect competitive ELISA were15ng/ml and0.1ng/ml respectively, whereas for the direct competitive, the IC50value and LOD were7.1ng/ml and0.04ng/ml respectively, the recoveries of COT were from97.23%to111.266%and coefficients of variation were from4.63%to12.9%.
For BPA, we developed and compared direct and indirect competitive ELISAs for the detection of BPA in human urine. The IC50value and LOD for the indirect competitive ELISA were7.0ng/ml and0.08ng/ml respectively, the recoveries of BPA were from100.99%to113.81%and coefficients of variation were from6.23%to14.58%, whereas for the direct competitive, the The IC50value and LOD for the indirect competitive ELISA were5.5ng/ml and0.03ng/ml respectively; the recoveries of BPA were from97.34%to119.42%and coefficients of variation were from7.37%to 15.46%.
The IC50value and LOD of Pb for indirect competitive ELISA were5.09ng/ml and0.01ng/ml respectively, the recoveries of Pb were from98.7%to108.3%and coefficients of variation were from2.83%to13.92%. The IC50value and LOD of Cd for indirect competitive ELISA were10.1ng/ml and0.07ng/ml respectively, the recoveries of Cd were from95.6%to103.9%and coefficients of variation were from2.56%to12.99%. The IC50value and LOD for the indirect competitive ELISA about Cu were7.3ng/ml and0.04ng/ml respectively, the recoveries of Cu were from93.2%to106.5%and coefficients of variation were from2.67%to14.23%.
Conclusions'. The results indicated that all the methods developed in this research were highly specific, highly sensitive and easy-to-use, and could be used for the detection of environmental pollutants in a large number of human samples.
2. Development of Immuno-PCR assay for detection of SAL
Background:It's necessary to develop a more sensitive method to detect the environmental pollutants with the constant improvement of human health needs.
Objectives:The aim of our study was to develop a novel pre-assembled DNA immunoprobe based direct competitive immuno-PCR for the determination of SAL level in human urine.
Methods:In this study, we focused on SAL, modified the traditional immuno-PCR, conjugated the analyte SAL to reporter DNA and for the first time we have developed a novel pre-assembled DNA immunoprobe based direct competitive immuno-PCR to investigate the level of small molecule SAL in human urine. There were only three steps in this assay, namely SAL antibody coating-competition between free SAL and DNA conjugated SAL-PCR amplification.
Results:Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, and the limit of detection of SAL in human urine was found to be21fg/ml, the recoveries of SAL were from85.5%to98.3%and coefficients of variation were from5.76%to9.23%.
Conclusions:This method combines the specificity of immunoassays with PCR signal amplification and provides a huge improvement of sensitivity of SAL. To the best of our knowledge, the sensitivity in this study is one of the highest sensitivity observed for detecting SAL. Moreover, this method can be modified and used for detecting other small molecule chemicals in environment and could lead the trend to the ultrasensitive detection of small molecules chemicals.
3. Determination of internal exposure of human to different environmental pollutants and preliminary correlation analysis between environmental pollutants and human disease
Objective:To understand the exposure to several common environmental pollutants in general population and preliminary study correlation between environmental pollutants and human disease.
Methods:Using the methods developed in this research, we measured the urinary concentrations of TAR, COT, SAL, Pb and AFM1in the selected participants. After detection, descriptive and correlation analysis were conducted by SPSS.
Results:The detection range for TAR in351participants was0-109.4ng/ml; the GM for COT in951participants was10.32ng/ml, the detection range was0-6257ng/ml; the GM for SAL in951participants was0.61ng/ml, the detection range was0-16.06ng/ml; the GM for Pb in951participants was0.23ng/ml, the detection range was0-139ng/ml. In the correlation analysis between environmental pollutants and human disease, we found higher urinary COT concentrations were associated with lung cancer.
Conclusions:All environmental pollutants in our research can be detected in the selected participants, and further studies are needed to reveal the effects of chronic exposure on human.
To sum up:
In our study, we explored two problems that limited the development of research about human exposure to environmental pollutants. To improve the detection throughput, we have established six different methods to detect human exposure to environmental pollutants based on ELISA. These methods have high sensitivity and specificity, more importantly, the throughput of detection has improved significantly. For the improvement of the sensitivity, we combined the high specificity of immunological and high sensitivity of the PCR reaction, and developed the modified immuno-PCR which is used specific to the detection of low molecular weight compound. The modified immuno-pcr was first applied to the detection of a low molecular weight compound (SAL) in our study. The method shows a super highly improvement to sensitivity and is found to be the most sensitive method to SAL detection. In addition, we also preliminary detected human exposure to several different environmental pollutants and analyzed the relationship between environmental pollutants and population health. This is a first systematic study about human exposure to environmental pollutants which from technical research to detection on human exposure to correlation analysis between the health and human exposure to pollutants. This study provided technical supports and theoretical basis for the generalization of detection about internal exposure, investigated the population distribution to environmental pollutants, established the corresponding primary database of human exposure level to environmental pollutants, and laid the foundation to the fomulation of limit value standard and correlation analysis between the human diseases and environmental pollutants.
引文
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