以多聚赖氨酸为载体的双酚A包被抗原制备及其免疫分析研究

发布时间：2018-08-15 13:38

【摘要】：双酚A (BPA)是一种内分泌干扰物,影响生殖系统的发育。双酚A还可导致前列腺癌、神经系统和胰腺功能混乱。双酚A是一种重要的有机化工原料,被用于生产环氧树脂与聚碳酸酯类产品,而这两类产品在食品包装、食具、塑料瓶等方面应用广泛。双酚A在一定条件下会因溶出、迁移和排放等原因进入生态环境,进而对人体健康造成潜在危害。为此,建立一种快速检测双酚A的方法具有十分重要的意义。本文利用双酚A结构类似物为半抗原,采用碳二亚胺法将双酚A半抗原与牛血清白蛋白(BSA)、人血清白蛋白(HSA)等载体偶联合成双酚A免疫抗原,再将双酚A半抗原与多聚赖氨酸(PLL)、鸡血清白蛋白(OVA)偶联制备包被抗原。用双酚A免疫抗原免疫新西兰兔,以制备其多克隆抗体。选取最佳的免疫抗原免疫Balb/c鼠,取其脾细胞与骨髓瘤细胞细胞SP2/0融合,经多次亚克隆筛选出高性能的单克隆细胞株。分别利用制备的多克隆抗体和单克隆抗体,建立了双酚A免疫分析方法。利用双酚A多克隆抗体,优化免疫分析条件,分别建立检测双酚A的酶联免疫分析吸附法和荧光免疫分析法。建立的酶联免疫分析方法线性范围为1.3ng/mL～292.2ng/mL,最低检测限是0.5ng/mL,样品加标回收率为86.7%-109.2%。用酶联免疫分析法研究西洋菜对双酚A的去除能力,结果表明对其去除效果较好。荧光免疫分析的的线性范围为2.4ng/mL～1279.5ng/mL,最低检测限是0.8ng/mL,样品回收率为89.3%-110.6%。由此可知,建立的酶联免疫分析方法的灵敏度更高。利用体内诱生腹水法制备双酚A单克隆抗体,优化免疫分析条件,建立检测双酚A的生物素-亲和素放大酶联免疫吸附法。其线性线性范围为0.9ng/mL～678.7ng/mL,最低检测限是0.3ng/mL,样品回收率为84.0%-106.0%。建立的生物素-亲和素放大体系具有更低的检测限与交叉反应,适于双酚A的痕量检测。
[Abstract]:Bisphenol A (BPA) is an endocrine disruptor that affects the development of the reproductive system. Bisphenol A can also lead to prostate cancer, nervous system and pancreatic dysfunction. Bisphenol A (BPA) is an important organic chemical raw material, which is used to produce epoxy resin and polycarbonate products, which are widely used in food packaging, food utensils, plastic bottles and so on. Under certain conditions, bisphenol A may enter the ecological environment due to its dissolution, migration and emission, which may cause potential harm to human health. Therefore, it is of great significance to establish a rapid method for the detection of bisphenol A. Bisphenol A immune antigen was synthesized by coupling bisphenol A hapten with bovine serum albumin (BSA),) human serum albumin (HSA) by carbodiimide method. Bisphenol A hapten was coupled with poly-lysine (PLL), chicken serum albumin (OVA) to prepare coated antigen. New Zealand rabbits were immunized with bisphenol A immune antigen to prepare polyclonal antibodies. Balb/c mice were immunized with the best immune antigen. The spleen cells were fused with myeloma cells (SP2/0) and the high performance monoclonal cell lines were selected by subcloning. An immunoassay method for bisphenol A was established by using polyclonal antibody and monoclonal antibody. The enzyme linked immunosorbent assay (Elisa) and fluorescence immunoassay (FIA) were established for the detection of bisphenol A by using polyclonal antibody of bisphenol A and optimizing the conditions of immunoassay. The linear range of enzyme-linked immunosorbent assay (Elisa) was 1.3ng / mL-1 292.2ng / mL, the lowest detection limit was 0.5ng / mL, and the recoveries of samples were 86.7% -109.2%. The removal ability of bisphenol A was studied by enzyme linked immunosorbent assay (Elisa). The linear range of fluorescence immunoassay is 2.4ng / mLN 1279.5ng / mL, the lowest detection limit is 0.8ng / mL, and the sample recovery is 89.3- 110.6. Therefore, the sensitivity of the established Elisa method is higher. The monoclonal antibody against bisphenol A was prepared by inducing ascites in vivo, and the immunoassay conditions were optimized. A biotin-affinity enzyme linked immunosorbent assay (Elisa) was established for the detection of bisphenol A (BPA) by enzyme linked immunosorbent assay (Elisa). The linear range is 0.9ng / mL ~ 678.7ng 路mL ~ (-1), the lowest detection limit is 0.3ng / mL, and the sample recovery is 84.0 ~ 106.0. The biotin-avidin amplification system has lower detection limit and cross reaction, which is suitable for the trace detection of bisphenol A.
【学位授予单位】：广东工业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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