几种典型环境内分泌干扰物的酶联免疫吸附分析方法研究
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摘要
环境内分泌干扰物是指由于人类的生产和生活活动而释放到环境中的、对人体内和动物体内原本营造的正常激素功能施加影响,从而影响内分泌系统的化学物质作用的物质,又称为“环境激素”。它是一类对生物体有害的化学物质,具有类似雌激素的作用,暴露到环境中可导致动物和人体的性激素分泌量及活性下降,精子数量减少,生殖器官异常,癌症等疾病的发病率增加,并使生殖能力降低,同时还影响到各种生物的免疫和神经系统,也可导致生物基因突变等。环境内分泌干扰物问题已经成为继温室效应、臭氧层破坏之后的第三大环境问题,对其筛选及检测方法的研究是国际上环境科学的研究前沿和热点之一。
本文主要研究了测定环境中萘(NA)、4,4'-二溴联苯(PBB15)、3,4-二氯联苯(PCB12)的酶联免疫吸附分析(enzyme-linkedimmunosorbent assay,ELISA)新方法,同时与传统的经典的色谱法-高效液相色潜检测结果进行相关性分析。
本文主要研究成果简述如下:
(1)利用本组制备的抗NA多克隆抗体和人工包被原,优化ELISA相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定NA的间接竞争酶联免疫吸附分析方法,其线性范围为10~(-3)-10μg/L,检出限IC_(20)为0.25ng/L,并将该法用于测定东华大学镜月湖湖水、学校生活区喷泉水和苏州河河水中NA的含量,回收实验显示回收率在87%-107%之间。这种方法灵敏度高、选择性好、线性范围宽,效果理想。同时用上述经优化的间接竞争ELISA法检测印染废水处理厂底泥中NA的含量,并与高效液相色谱检测结果作相关性分析,得出两者相关系数为0.9867,可见两种方法的检测结果数值比较接近。
(2)利用本组制备的PBB15多克隆抗体和自己制备的PBB15人工包被原,优化相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定PBB15的间接竞争酶联免疫吸附分析方法,其线性范围在10~(-2)-10~2μg/L,检测限IC_(20)为0.02μg/L,并将该法用于测定东华大学镜月湖湖水、学校生活区喷泉水和苏州河河水中的PBB15,回收实验表明回收率在97%-147%之间,同时用上述经优化的间接竞争ELISA法测定印染废水处理厂底泥中PBB15的含量,并与高效液相色谱检测结果作相关性分析,得出两者相关系数为0.9048。
(3)利用本组制备的PCB12多克隆抗体和自己制备的PCB12人工包被原,经测定,其抗体效价为100,优化ELISA相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定PCB12的间接竞争ELISA方法,其线性范围在10~(-2)-10~2μg/L,检测限IC_(20)为4.04ng/L,并将该法用于测定茶叶中PCB12的含量,回收率在95%-113%之间,同时用高效液相色谱检测茶叶中PCB12含量,将两者结果做相关性分析,得出相关系数为0.9894,可见ELISA的检测结果比较可靠。
ELISA的检测限明显低于HPLC,能满足环境中微量甚至痕量物质的检测要求,但是线性相关性没有HPLC好。HPLC的线性范围比ELISA要宽几个数量级,因此在用HPLC检测微量甚至痕量物质时需要进行浓缩富集,由此大大加大了检测的成本和时间,而ELISA的建立突破了这个瓶颈。
Environmental endocrine disruptors, also called environmental hormones, act as estrogensin humans and animal bodies. Most of them are discharged into the environment with theproduction and activities of the humanity. It has been found that environmental endocrinedisruptors have adverse effects on endocrine system in humans and animals, once exposed to theenvironment can lead to less secretion of sex hormones and poorer activity in humans and animals,more diseases like azoospermia, hypo genesis of reproductive system and cancer, effecting onimpair fertility, immune system and nervous system, causing gene mutation. Environmentalendocrine disruptors have become the third environmental problem after the global wanning andozonosphere destruction. Now the research on screening and determining the environmentalendocrine disruptors has become one of the hotpots of environmental science.
In this study, we researched on new ELISA methods for detecting Naphthalene (NA) , p,p'-Dibromobiphenyl (PBB15) and 3,4 -Dichlorobiphenyl (PCB12) , respectively. Meanwhile,compared the results detected by ELISA and HPLC, made a correlation analysis between them.
(Ⅰ) ELISA method was developed for the analysis of NA in the Jingyue Lake, fountain andSuzhou River, with the antibody and coating antigen made by our lab. For the optimum conditionsof experiment,such as pH, sodium chloride and methanol content in PBS, blocking agents,washing solution as well as concentration of goat anti-rabbit HRP-IgG, the linear calibration rangewas 10~(-3)-10μg/L, the detection limit IC_(20) was 0.2500 ng/L. Spiked recoveries from three watersamples were 87%-107% for NA from ELISA which was satisfactory. Meanwhile, the linearrelationship between ELISA and HPLC measurements of the dyeing sludge sample was strong inthe combined data, the correlation coefficient reached 0.9867.
(Ⅱ) ELISA method was developed that can used for the detection of PBB15 in the JingyueLake, fountain and Suzhou River with the antibody made by our lab, and the coating antigen madeby me. Under optimized assay conditions, the linear calibration range was 10~(-2)-10~2μg/L, thedetection limit IC_(20) was 0.02000μg/L. Spiked recoveries from three water samples were97%-147% for PBB15 from ELISA which was satisfactory. Meanwhile, the correlation of theresults detected both by ELISA and HPLC in the dyeing sludge sample was well, both methodswere highly correlated (r = 0.9048).
(Ⅲ) ELISA method was developed for the analysis of PCB12 in the tea with the antibodymade by our lab and the coating antigen made by me. Titer for determination of antibody byindirect ELISA was 100. Under optimized assay conditions, the linear calibration range was 10~(-2)-10~2 ug/L, the detection limit IC_(20)was 4.040 ng/L. Spiked recoveries from tea sample were95% -113% for PCB12 from ELISA which was satisfactory. Meanwhile, the correlation of theresults detected by ELISA and HPLC in the tea sample acted well, the correlation coefficient was0.9894.
Compared with HPLC, the detection of limit for ELISA was lower, which can meet thetesting requirements of trace materials existing in environment. The line range of HPLC is widerthan ELISA, therefore the analytes have to be enriched, it causes incremental cost andtime-extension.However, the correlation and repeatability of ELISA are not so good as HPLC.
本文主要研究了测定环境中萘(NA)、4,4'-二溴联苯(PBB15)、3,4-二氯联苯(PCB12)的酶联免疫吸附分析(enzyme-linkedimmunosorbent assay,ELISA)新方法,同时与传统的经典的色谱法-高效液相色潜检测结果进行相关性分析。
本文主要研究成果简述如下:
(1)利用本组制备的抗NA多克隆抗体和人工包被原,优化ELISA相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定NA的间接竞争酶联免疫吸附分析方法,其线性范围为10~(-3)-10μg/L,检出限IC_(20)为0.25ng/L,并将该法用于测定东华大学镜月湖湖水、学校生活区喷泉水和苏州河河水中NA的含量,回收实验显示回收率在87%-107%之间。这种方法灵敏度高、选择性好、线性范围宽,效果理想。同时用上述经优化的间接竞争ELISA法检测印染废水处理厂底泥中NA的含量,并与高效液相色谱检测结果作相关性分析,得出两者相关系数为0.9867,可见两种方法的检测结果数值比较接近。
(2)利用本组制备的PBB15多克隆抗体和自己制备的PBB15人工包被原,优化相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定PBB15的间接竞争酶联免疫吸附分析方法,其线性范围在10~(-2)-10~2μg/L,检测限IC_(20)为0.02μg/L,并将该法用于测定东华大学镜月湖湖水、学校生活区喷泉水和苏州河河水中的PBB15,回收实验表明回收率在97%-147%之间,同时用上述经优化的间接竞争ELISA法测定印染废水处理厂底泥中PBB15的含量,并与高效液相色谱检测结果作相关性分析,得出两者相关系数为0.9048。
(3)利用本组制备的PCB12多克隆抗体和自己制备的PCB12人工包被原,经测定,其抗体效价为100,优化ELISA相关反应条件后,如PBS的pH值、NaCl浓度、甲醇含量、洗涤液、封闭液、酶标二抗的稀释倍数等,建立了测定PCB12的间接竞争ELISA方法,其线性范围在10~(-2)-10~2μg/L,检测限IC_(20)为4.04ng/L,并将该法用于测定茶叶中PCB12的含量,回收率在95%-113%之间,同时用高效液相色谱检测茶叶中PCB12含量,将两者结果做相关性分析,得出相关系数为0.9894,可见ELISA的检测结果比较可靠。
ELISA的检测限明显低于HPLC,能满足环境中微量甚至痕量物质的检测要求,但是线性相关性没有HPLC好。HPLC的线性范围比ELISA要宽几个数量级,因此在用HPLC检测微量甚至痕量物质时需要进行浓缩富集,由此大大加大了检测的成本和时间,而ELISA的建立突破了这个瓶颈。
Environmental endocrine disruptors, also called environmental hormones, act as estrogensin humans and animal bodies. Most of them are discharged into the environment with theproduction and activities of the humanity. It has been found that environmental endocrinedisruptors have adverse effects on endocrine system in humans and animals, once exposed to theenvironment can lead to less secretion of sex hormones and poorer activity in humans and animals,more diseases like azoospermia, hypo genesis of reproductive system and cancer, effecting onimpair fertility, immune system and nervous system, causing gene mutation. Environmentalendocrine disruptors have become the third environmental problem after the global wanning andozonosphere destruction. Now the research on screening and determining the environmentalendocrine disruptors has become one of the hotpots of environmental science.
In this study, we researched on new ELISA methods for detecting Naphthalene (NA) , p,p'-Dibromobiphenyl (PBB15) and 3,4 -Dichlorobiphenyl (PCB12) , respectively. Meanwhile,compared the results detected by ELISA and HPLC, made a correlation analysis between them.
(Ⅰ) ELISA method was developed for the analysis of NA in the Jingyue Lake, fountain andSuzhou River, with the antibody and coating antigen made by our lab. For the optimum conditionsof experiment,such as pH, sodium chloride and methanol content in PBS, blocking agents,washing solution as well as concentration of goat anti-rabbit HRP-IgG, the linear calibration rangewas 10~(-3)-10μg/L, the detection limit IC_(20) was 0.2500 ng/L. Spiked recoveries from three watersamples were 87%-107% for NA from ELISA which was satisfactory. Meanwhile, the linearrelationship between ELISA and HPLC measurements of the dyeing sludge sample was strong inthe combined data, the correlation coefficient reached 0.9867.
(Ⅱ) ELISA method was developed that can used for the detection of PBB15 in the JingyueLake, fountain and Suzhou River with the antibody made by our lab, and the coating antigen madeby me. Under optimized assay conditions, the linear calibration range was 10~(-2)-10~2μg/L, thedetection limit IC_(20) was 0.02000μg/L. Spiked recoveries from three water samples were97%-147% for PBB15 from ELISA which was satisfactory. Meanwhile, the correlation of theresults detected both by ELISA and HPLC in the dyeing sludge sample was well, both methodswere highly correlated (r = 0.9048).
(Ⅲ) ELISA method was developed for the analysis of PCB12 in the tea with the antibodymade by our lab and the coating antigen made by me. Titer for determination of antibody byindirect ELISA was 100. Under optimized assay conditions, the linear calibration range was 10~(-2)-10~2 ug/L, the detection limit IC_(20)was 4.040 ng/L. Spiked recoveries from tea sample were95% -113% for PCB12 from ELISA which was satisfactory. Meanwhile, the correlation of theresults detected by ELISA and HPLC in the tea sample acted well, the correlation coefficient was0.9894.
Compared with HPLC, the detection of limit for ELISA was lower, which can meet thetesting requirements of trace materials existing in environment. The line range of HPLC is widerthan ELISA, therefore the analytes have to be enriched, it causes incremental cost andtime-extension.However, the correlation and repeatability of ELISA are not so good as HPLC.
引文
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