【作者】 李畅；

【导师】 柳增善；

【作者基本信息】 吉林大学 ， 兽医（专业学位）， 2018， 硕士

【摘要】 邻苯二甲酸二(2-乙基)己酯(DEHP),属于邻苯二甲酸酯类物质(PAEs),是一种小分子类环境激素。DEHP是PAEs中生产量和使用量最大的一类物质,添加在聚乙烯塑料产品中有增强柔韧性的作用,目前有很大的商业利用价值,一般应用于食品包装等多种塑料产品中,与我们的生活息息相关。虽然这类物质呈中等毒性,但是在环境中性质比较稳定,不易分解,随食品等进入人体后参与新陈代谢,其代谢产物单酯的毒性明显强于其原型本身。若长期暴露于DEHP含量较高的环境中,产生的毒性累积效应轻则导致血细胞减少,重则导致肝癌和肾功能不全,可激发潜在的癌细胞和致突变性,并造成内分泌失调,胎儿畸形,幼儿性别错乱以及生殖系统功能丧失。目前国标上针对DEHP的检测方法主要是仪器检测法,但比较繁琐和经济代价较高。而近些年来免疫学检测也逐渐应用到商品中小分子化学物质的大规模检测,如常见的ELISA试剂盒。而ELISA检测法与液相色谱法等相比具备原理简单、简便易行、价格低廉、灵敏并且适用于大批量样品筛选等优点,所有的操作始终围绕ELISA方法的特异敏感优势,即抗原抗体之间的特异性敏感性反应。所以ELISA方法在PAEs检测中逐渐得到应用。本实验首先根据半抗原设计的基本原则,设计出在DEHP结构基础上增加3个碳臂的半抗原结构(DBCP),制备完全免疫抗原DEHP-BSA来免疫小鼠,获得抗DEHP血清,制备检测抗原DEHP-OVA作为包被抗原,利用目标检测物与微孔板中抗原竞争结合DEHP多克隆抗体的原理建立检测DEHP的IC-ELISA方法;并且同时将检测抗原DEHP-OVA与羧基磁珠偶联制备磁珠探针,本实验分别建立了间接竞争ELISA(IC-ELISA)方法和基于纳米磁珠的DEHP间接竞争ELISA(IMB-IC-ELISA)方法两种方法进行实际食品样品中DEHP的检测。首先利用混合酸酐法将小分子DEHP与卵清白蛋白(OVA)偶联制备检测抗原(DEHP-OVA),建立间接竞争性ELISA(IC-ELISA),之后对主要条件进行优化,优化之后的主要步骤为:检测抗原工作浓度为2μg/m L,包被时间为37℃,4 h;最佳封闭条件为1%脱脂奶粉,37℃,0.5 h;抗体最佳稀释倍数及工作条件为1:16000,37℃,1 h;二抗最佳稀释倍数及工作条件:1:10000,37℃,1 h;TMB底物最佳显色条件:37℃,15 min;硫酸终止2 min后读值。得到线性回归方程为:y=69.873 x-79.811,R2=0.9945,IC50为63.19 ng/m L,该方法检测范围16.91-236.11 ng/m L;对阴性水和饮料样品、阴性油样和阴性酒样,分别添加浓度为200 ng/m L、62.5 ng/m L、31.25 ng/m L的标准品模拟实际样品,水和饮料样品对应的回收率分别为116.51%、112.99%、112.06%;油样对应的回收率分别为110.70%、102.84%、94.20%;油样对应的回收率分别为91.84%、92.26%、95.12%;从超市购买样品105份,根据标准曲线,测出实际样品中DEHP含量,并以国标法检测结果作对照。将检测抗原DEHP-OVA与羧基磁珠偶联制备磁珠探针,在磁场的作用下,相当于包被检测抗原,按照普通IC-ELISA原理,建立IMB-IC-ELISA检测方法,对主要条件进行优化,IMB-IC-ELISA检测方法最佳操作步骤为:磁珠探针最佳用量为60μg,多抗最佳稀释倍数及工作条件:1:4000,37℃孵育1 h;二抗稀释倍数及工作条件:1:6000,37℃孵育40 min;TMB底物最佳显色条件:37℃孵育10 min;硫酸终止2 min后读值。获得线性回归方程:y=68.482x-112.61,R2=0.9924,该检测方法对DEHP的检测范围是58.16 ng/m L-824.15ng/m L;IC50=218.94 ng/m L。模拟实际样品添加浓度为500 ng/m L、250 ng/m L、100 ng/m L的标准品,计算实际检测浓度,对应的回收率分别为90.46%、92.38%、89.12%;将采集的酒类样品以IMB-IC-ELISA法进行检测,根据建立的标准曲线计算出相应的DEHP含量,同时以国标法检测结果作对照。结果显示,所检测样品符合国家检测标准,本实验建立的IC-ELISA方法检测结果和IMB-IC-ELISA结果与国标法GC-MS结果基本一致,且结果相比较于普通IC-ELISA,IMB-IC-ELISA方法耗时更短,成本较高,结果与国标法结果更加接近。更多还原

【Abstract】 Di(2-ethylhexyl)phthalate(DEHP),is a kind of phthalate esters(PAEs)and is commonly known as environmental hormone.DEHP is a type of plasticer with the largest amount of production and usage in PAEs.It is used to enhance flexibility in polyethylene plastic products and has great commercial utility value.It is generally used in hundreds of cosmetics,food packaging,and pesticides.The harm of long-term exposure to high levels of DEHP are as follows:the toxicity of its monoester metabolites was significantly stronger than itself and the cumulative effect of toxicity is caused by a decrease in blood cells,which leads to liver cancer and renal insufficiency and can cause endocrine disorders,fetal malformations,young children’s gender disorders,and loss of reproductive system function.At present,the national standard detection method for DEHP detection method mainly includes instrument detection method.In recent years,immunological testing has gradually been applied to large-scale detection of small and medium-sized chemical substances in commodities,such as common ELISA kits and colloidal gold test strips.The ELISA method has the advantages of simple principle,simplicity,low cost,sensitivity,and suitability for large-scale sample detection.Therefore,the ELISA method can be used to detect PAEs.According to the basic principle of hapten design,a hapten structure(DBCP)that meets the requirements of adding two carbon arms to the structure of DEHP was designed.Anti-DEHP serum was prepared by immunizing mice with DEHP-BSA.DEHP-OVA was used as a coating antigen to develop an IC-ELISA method for detecting DEHP.At the same time,DEHP-OVA was coupled with a carboxyl magnetic bead to prepare a magnetic bead probe.An indirect competition ELISA(IC-ELISA)method and DEHP indirect competition ELISA(IMB-IC-ELISA)basedon magnetic beads were developed,were used to detect DEHP in liquid food samples.A solid phase detection antigen(DEHP-OVA)was prepared by coupling a small molecule of DEHP with ovalbumin(OVA)using a mixed anhydride method.The indirect detection principle was that the target analyte and the solid phase antigen in the microtiter plate competed with the specific polyclonal antibody.Competitive ELISA(IC-ELISA).After the optimization of the main conditions of IC-ELISA detection,the main steps after optimization are as follows: concentration of detection antigen is 2 μg/m L,coating condition is 37℃,4 h or 4℃,12 h incubation;blocked condition is 1% skim milk powder,37℃,0.5 h;The optimal dilution of antibody and working conditions is 1: 16000,37℃,1 h;The optimal dilution of HRP Goat anti-mice antibody and working conditions is 1: 10000,37℃,1 h;TMB substrate coloring solution 37℃,15 min;H2SO4 solution terminated.The linear regression equation was: y=69.873x-79.811,R2=0.9945,IC50=63.19 ng/m L,the method detection range was 16.91-236.11 ng/m L;Adding DEHP 200.00 ng/m L,62.50ng/m L,and 31.25 ng/m L respectively to negative water and beverage samples,negative oil samples,and negative wine samples to get the corresponding recoveries for water and beverage samples were respectively 116.51%,112.99%,and 112.06%;the corresponding recoveries for the oil samples were 110.70%,102.84%,94.20%;the corresponding recovery rates of oil samples were 91.84%,92.26%,and 95.12%respectively;105 pieces of samples were purchased from the supermarket.According to the standard curve,the content of DEHP in the actual samples was measured.The detection antigen DEHP-OVA is coupled with a carboxyl magnetic bead to prepare a magnetic bead probe to used as a detection antigen,under the action of a magnetic field,it is equivalent to a coating step.The principle of the IMB-IC-ELISA is the same as that of an ordinary IC-ELISA.Methods The main conditions were optimized.The best procedure for IMB-IC-ELISA were as follows: the optimal amount of magnetic bead probe was 60 μg,the optimal dilution of antibody and the working condition was 1:4000,incubated for 1 hour at 37℃;The HRP Goatanti-mice antibody dilution and working conditions were 1:6000,incubated at 37℃ for 40 min;TMB substrate solution was incubated at 37℃ for 10 min,H2SO4 solution terminated.The linear regression equation of DEHP is: y=68.482x-112.61,R2=0.9924.The detection range of DEHP is 58.16-824.15 ng/m L;IC50=218.94ng/m L.The actual samples were spiked with DEHP 500 ng/m L,250 ng/m L,and 100ng/m L.DEHP content was calculated according to the standard curve established.The results showed that the all samples met the national testing standards.The results of the IC-ELISA and IMB-IC-ELISA developed in this experiment were basically consistent with the results of the GC-MS method.Compared with IC-ELISA,IMB-IC-ELISA method has a higher cost,and the result is closer to the national standard method result.更多还原