【作者】 李灼坤；

【导师】 过世东； 胥传来；

【作者基本信息】 江南大学 ， 水产品加工及贮藏工程， 2013， 博士

【摘要】 随着毒理学研究的不断深入,合成色素和雌激素效应物的毒性作用己逐渐被人们认识。然而,食品中合成色素违法添加和雌激素效应物超标的食品安全事件不断出现,引起了研究者和消费者的共同关注。目前,我国在食品安全监管方面缺乏简便、快速、灵敏、准确检测这些合成色素和雌激素效应物的方法。基于此,本课题采用免疫学原理,在制备特异性单克隆抗体的基础上,建立起赤藓红、柠檬黄、碱性橙Ⅱ、对氨基偶氮苯、辛基酚和邻苯二甲酸二丁酯六种物质的ELISA检测方法,主要研究内容如下：(1)通过将赤藓红脱钠后与6-氨基己酸反应合成赤藓红的半抗原,并合成赤藓红的人工完全抗原,免疫老鼠得到针对赤藓红的阳性脾细胞,进行细胞融合并获得赤藓红的单克隆细胞株,通过体内诱生法制备腹水单抗,从而建立赤藓红的酶联免疫检测方法,赤藓红单抗的IC50为3.02ng/mL, LOD检测限(IC1o)为0.75ng/mL,线性范围为1.27ng/mL-8.49ng/mL。将赤藓红的ELISA检测方法成功应用到葡萄汁饮料的添加回收实验当中,其批内批间测定的总回收率为92.06%-106.20%,总的变异系数为4.38%-13.93%。(2)研究了影响制备高特异性高灵敏柠檬黄抗体的半抗原因素机理,并合成了相关的半抗原,最终免疫筛选出一株超高灵敏的柠檬黄单克隆抗体1F3,其IC50值达到了0.11ng/mL。并用建立的ELISA方法对橙汁饮料进行了添加回收实验,其批内的回收率为84.12%-109.70%,变异系数为5.95%-11.26%；批间的回收率为73.38%-93.43%,变异系数为5.90%-12.35%。并且还将该ELISA方法成功地应用到了真实未知的阳性碳酸饮料样品的检测,并评价了其精确性,其检出浓度值为13456.73ng/mL。(3)通过对碱性橙Ⅱ的化学结构进行分析,设计合成出带有4个C原子的羧基碳链的碱性橙类似物,并解决了碱性橙分子本身所具有的氨基的保护间题,制备出高特异性的碱性橙单克隆抗体5E12,其最优IC5o为2.13ng/mL,检测限LOD为0.50ng/mL,线性范围在0.81-6.93ng/mL。并用建立的ELISA检测方法对市售的小黄鱼进行碱性橙添加回收测定,其总回收率为75.52%-112.95%,总变异系数为4.99%-7.96%,证明该方法灵敏稳定可靠。(4)以对氨基苯甲酸和间苯二胺进行重氮化反应,制备出了对氨基偶氮苯的半抗原,并免疫老鼠和进行细胞融合,制备筛选出对氨基偶氮苯的单克隆抗体1G3,其最优IC50达到了44.11ng/mL,检测限LOD (IC10)为5.09ng/mL,且其对碱性橙等其它色素均没有交叉反应。用1G3抗体的ELISA方法对豆腐干进行了实际样品添加回收实验,其总回收率为76.17%-88.26%,总变异系数为3.75%-13.30%,可用于食品中对氨基偶氮苯非法添加物的快速灵敏检测。(5)在前人研究的基础上,以壬二酸和壬二酸二甲酯为原料,通过6步化学反应来合成辛基酚的类似物壬酸苯酚NPA,制备出了具有更强特异性的只针对辛基酚的单克隆抗体3C7,其IC50为75.83ng/mL,检测限LOD (IC10)为6.55ng/mL,并且该单抗对壬基酚等其余酚类物质无交叉反应。用该单抗建立的ELISA检测方法,对太湖水进行了添加回收实验,其总体回收率为79.53%-96.47%,变异系数为4.79%-12.31%。(6)针对目前酞酸酯免疫检测方法中邻苯二甲酸二丁酯DBP的抗体灵敏度不高的间题,设计合成了以4-氨基邻苯二甲酸二丁酯DBAP作为半抗原,进一步制备筛选出针对邻苯二甲酸二丁酯DBP的高特异性单克隆抗体3B7,其IC50达到了33.94ng/mL,检测限LOD为3.31ng/mL,且对其余酞酸酯无交叉反应。将建立的DBP单抗ELISA检测方法用于酒类的添加回收实验中,其对市售的白酒实际样品的回收率在80.3%-95.1%之间。更多还原

【Abstract】 With the deepening of toxicology studies, the toxic effects of synthetic pigments and estrogeniceffectors have gradually been recognized. However, food safety incidents about the excessive andillegal addition of synthetic pigments and estrogenic effectors in foods, continue to occur, whichhave attracted much attentions from researchers and consumers. At present, there are still lack ofsimple, rapid, sensitive and accurate detection methods in terms of China’s food safety supervisionfor these synthetic pigments and estrogenic effectors. Therefore, in this project, we produced highspecific monoclonal antibodies and established ELISA methods for the detection of six substancesincluding erythrosine, tartrazine, basic orange II,4-aminoazobenzene, octylphenol anddibutyl-o-phthalate, based on the principles of immunology. The main contents are as follows:A new and innovative erythrosine hapten was achieved by removing the sodium fromerythrosine and coupling with6-aminocaproic acid. The hapten was conjugated to carrier proteinsto give the artificial completed antigens of erythrosine. BALB/c mice were immunized by theimmunogen to produce the positive spleen cells which could secrete antibodies against erythrosine.The erythrosine monoclonal cell lines were obtained after cells fusion of spleen cells and hybridomacells.The ascites monoclonal antibody was collected from the mice using in vivo induction method,in order to establish erythrosine ELISA method. The IC50of erythrosine monoclonal antibody was3.02ng/mL, with a limit of detection (LOD, IC10) of0.75ng/mL, and the linear range was ranged1.27ng/mL to8.49ng/mL. The erythrosine ELISA method was successfully applied to thedetection of grape juice drinks sample through recovery test, in which the measured intra-assay andinter total recovery was all in the range of92.06%-106.20%and the overall coefficient of variationwas in the range of4.38%-13.93%.A study was carried out in order to investigate the influence of haptens on the production ofhigh specific and sensitive antibodies against tartrazine. Through the synthesis of haptens and theimmunization and so on, a super sensitive tartrazine monoclonal antibody1F3was finally produced,and its IC50value was as low as0.11ng/mL. The established ELISA method was further applied tothe recovery experiment of tartrazine in orange juice beverages. The intra-assay recovery rangedfrom84.12%to109.70%, and its coefficient of variation ranged from5.95%to11.26%; while theinter-assay recovery was from73.38%to93.43%, with its coefficient of variation from5.90%to12.35%. Futher more, in order to evaluate the accuracy of this ELISA method, this assay wassuccessfully applied to the detection of an unknown real positive carbonated sample, and theconcentration of this carbonated sample was13456.73ng/mL.Through the analysis of the chemical structure of basic orange II, a new analogue of basicorange II with a four carbon-atom carboxylated chain was designed and synthesized. And the problem of the protection of the amino in the basic orange molecule was also successfully sovled.Finally, the basic orange monoclonal antibody5E12with high specificity was successfully prepared.The optimized IC50was2.13ng/mL, with a limit detection (LOD) of0.50ng/mL. The linear rangewas0.81-6.93ng/mL. The established ELISA method was used for the detection of basic orange IIin commercially available small yellow croaker. The recovery test showed that the total recoveryranged75.52%to112.95%with the total coefficient of variation of4.99%-7.96%, which provedthat the method is very sensitive and reliable.A hapten of aniline yellow was prepared through diazotization reaction between aminobenzoicacid and metaphenylene diamine. The hapten was further used for the immunization of mice andcell fusion. A monoclonal antibody1G3of aniline yellow was produced. Its IC50value was as lowas44.11ng/mL and its LOD (IC10) was5.09ng/mL. There was no cross reaction between basicorange II and other pigments. The ELISA method based on the monoclonal antibody1G3wasemployed to analyze beancurd in a fortification experiment. The total recovery ranged from76.17%to88.26%, and the coefficient of variation ranged from3.75%to13.30%. This indicatd that thismethod can be applied to the rapid and sensitive detection of aminoazobenzene which wasforbidden to add to foods.A similar analogue (named NPA) of nonylphenol was synthesized through six-step chemicalreactions from azelaic acid and azelaic acid dimethyl ester, based on the previous studies. Amonoclonal antibody3C7with high specificity to octylphenol was produced. The IC50was75.83ng/mL and the LOD was6.55ng/mL. And this antibody against octylphenol had no crossreactivities to other phenols. A recovery experiment with water samples from Taihu Lake wasperformed with the ELISA method based on the monoclonal antibody.The total recovery was in therange of79.53%-96.47%, and the coefficient of variation was between4.79%-12.31%.In the current immunoassays for the dection of phthalate ester, the sensitivity of DBP antibodywas still not high. Therefore, the hapten DBAP was successfully synthesized, and a monoclonalantibody3B7against DBP was sucessfully prepared. The IC50of the antibody was33.94ng/mL andthe detection limit (LOD) was3.31ng/mL, no cross-reactivity was observed to the rest of otherphthalates. The ELISA method based on DBP monoclonal antibody was established for thefortification experiment in commercially available alcohol samples with the recovery of80.3%-95.1%.更多还原