黃麴毒素M1單株抗體之製備及其應用於酵素免疫分析法和奈米金粒子免疫層析試紙之開發

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/12224

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题名:	黃麴毒素M1單株抗體之製備及其應用於酵素免疫分析法和奈米金粒子免疫層析試紙之開發 Production of monoclonal antibody and development of ELISA and nanoparticle immunochromatographic strip assay for aflatoxin M1
作者:	朱光駿 Chu, Kuang-Chun
贡献者:	中山醫學大學:生物醫學科學學系碩士班;余豐益
关键词:	黃麴毒素M1;酵素免疫分析法;單株抗體;免疫層析試紙 aflatoxin M1;ELISA;monoclonal antibody;immunochromatographic strip
日期:	2015
上传时间:	2015-09-21T03:02:12Z (UTC)
摘要:	黴菌毒素普遍存在於日常生活之中，若不慎誤食到這些黴菌毒素會對人體造成危害。黃麴毒素M1 (AFM1)是動物食用被黃麴毒素B1汙染的飼料後，經動物體內羥化代謝後產生的產物容易殘存在動物的乳汁當中。本研究希望能夠開發一套靈敏又快速的檢測方式來檢測乳品中的AFM1。我們以AFM1毒素做為免疫抗原，並且以此一接合物來免疫小鼠，之後取其脾臟細胞與骨髓瘤細胞融合，最後篩選出融合瘤細胞株 (10F3C10)，分泌對AFM1具有專一性的單株抗體，並利用此一抗體來開發出檢測AFM1的直接競爭型酵素免疫分析法(Competitive direct enzyme-linked immunosorbent assay，cdELISA)。結果我們得到其抑制50％抗原-酵素接合物與抗體結合所需的抗原濃度(IC50)為 0.055 ng/mL。此外為了能夠現場快速檢測，本研究也利用AFM1的單株抗體與奈米金粒子相接合，進而開發一套更簡便且適用於一般大眾的快速免疫層析試紙(immunochromatographic strip assay)檢驗方法，此試紙的最低偵測含量為0.5 ~ 1 ng/mL 之間。本研究所開發的ELISA及免疫層析試紙具有高靈敏度及專一性並且能夠快速的檢測樣品中的AFM1毒素。 Mycotoxins are fugal secondary metabolites which are commonly found in our life. Human accidentally ingested these mycotoxins will harm the human health. Aflatoxin M1 (AFM1), a hydroxylated metabolite of aflatoxin B1, is often found in milk from animals fed with aflatoxin B1 contaminated feeds. This study focused on the development of sensitive and rapid methods to detect AFM1 levels in milk. Monoclonal antibodies specific for AFM1 are generated from mice after the animals have been immunized with AFM1-BSA. A monoclonal antibody (mAb) specific to AFM1 was produced from a stable hybridoma cell line, 10F3C10, which was generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from Balb/c mouse. The competitive direct enzyme-linked immunosorbent assay (cdELISA) was used for the characterization of the mAb for AFM1. The concentration leaves causing 50% inhibition (IC50) of binding of AFB1-horseradish peroxidase (AFB1-HRP) to the antibody by AFM1 was found to be 0.055 ng/mL. The detection level form the immunochromatographic strip assay, which was generated by conjugating mAb with gold nanoparticle, is 0.5 ~ 1 ng/mL for AFM1. In this study, both of the cdELISA and immunochromatographic strip can be a rapid and efficient way to detect the AFM1 in milk samples.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/12224
显示于类别:	[生物醫學科學學系暨碩士班] 博碩士論文

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