以多聚賴氨酸為載體的雙酚A包被抗原制備及其免疫分析研究
發(fā)布時(shí)間：2018-08-15 13:38
【摘要】：雙酚A (BPA)是一種內(nèi)分泌干擾物,影響生殖系統(tǒng)的發(fā)育。雙酚A還可導(dǎo)致前列腺癌、神經(jīng)系統(tǒng)和胰腺功能混亂。雙酚A是一種重要的有機(jī)化工原料,被用于生產(chǎn)環(huán)氧樹脂與聚碳酸酯類產(chǎn)品,而這兩類產(chǎn)品在食品包裝、食具、塑料瓶等方面應(yīng)用廣泛。雙酚A在一定條件下會因溶出、遷移和排放等原因進(jìn)入生態(tài)環(huán)境,進(jìn)而對人體健康造成潛在危害。為此,建立一種快速檢測雙酚A的方法具有十分重要的意義。 本文利用雙酚A結(jié)構(gòu)類似物為半抗原,采用碳二亞胺法將雙酚A半抗原與牛血清白蛋白(BSA)、人血清白蛋白(HSA)等載體偶聯(lián)合成雙酚A免疫抗原,再將雙酚A半抗原與多聚賴氨酸(PLL)、雞血清白蛋白(OVA)偶聯(lián)制備包被抗原。用雙酚A免疫抗原免疫新西蘭兔,以制備其多克隆抗體。選取最佳的免疫抗原免疫Balb/c鼠,取其脾細(xì)胞與骨髓瘤細(xì)胞細(xì)胞SP2/0融合,經(jīng)多次亞克隆篩選出高性能的單克隆細(xì)胞株。分別利用制備的多克隆抗體和單克隆抗體,建立了雙酚A免疫分析方法。 利用雙酚A多克隆抗體,優(yōu)化免疫分析條件,分別建立檢測雙酚A的酶聯(lián)免疫分析吸附法和熒光免疫分析法。建立的酶聯(lián)免疫分析方法線性范圍為1.3ng/mL～292.2ng/mL,最低檢測限是0.5ng/mL,樣品加標(biāo)回收率為86.7%-109.2%。用酶聯(lián)免疫分析法研究西洋菜對雙酚A的去除能力,結(jié)果表明對其去除效果較好。熒光免疫分析的的線性范圍為2.4ng/mL～1279.5ng/mL,最低檢測限是0.8ng/mL,樣品回收率為89.3%-110.6%。由此可知,建立的酶聯(lián)免疫分析方法的靈敏度更高。 利用體內(nèi)誘生腹水法制備雙酚A單克隆抗體,優(yōu)化免疫分析條件,建立檢測雙酚A的生物素-親和素放大酶聯(lián)免疫吸附法。其線性線性范圍為0.9ng/mL～678.7ng/mL,最低檢測限是0.3ng/mL,樣品回收率為84.0%-106.0%。建立的生物素-親和素放大體系具有更低的檢測限與交叉反應(yīng),適于雙酚A的痕量檢測。
[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.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392
本文編號：2184366
[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.
【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392
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