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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 微生物暨公共衛生學研究所 > 依資料類型分類 > 碩博士論文 >  豬瘟病毒醣蛋白Erns之單株抗體的製備及其應用於間接三明治ELISA抗體檢測方法之建立

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/154125

標題: 豬瘟病毒醣蛋白Erns之單株抗體的製備及其應用於間接三明治ELISA抗體檢測方法之建立
Preparation of the monoclonal antibody against classical swine fever virus glycoprotein Erns and its application to an indirect sandwich ELISA
作者: 黃靜如
Huang, Jing-Ju
Contributors: 黃千衿
Chienjin Huang
微生物暨公共衛生學研究所
關鍵字: 間接三名治ELISA;豬瘟病毒;單株抗體
indirect sandwich ELISA;classical swine fever virus;Erns;monoclonal antibody
日期: 2012
Issue Date: 2013-11-19 12:51:11 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 豬瘟(classical swine fever, CSF)是由豬瘟病毒(classical swine fever virus, CSFV)所引起之豬隻高度傳染性疾病。豬瘟病毒在分類上屬於黃病毒科(Flaviviridae)中之瘟疫病毒屬(Pestivirus)成員,而患病之豬隻有發燒、出血及淋巴球低下等主徵。而CSFV之醣蛋白Erns具有獨特之RNase活性亦可誘發宿主之抗體反應,故在致病機轉上扮演重要角色。本研究之目的為製備抗CSFV Erns之單株抗體(monoclonal antibody, Mab),並進一步應用做為血清學診斷工具。所得三株抗CSFV Erns單株抗體分別命名為1-41、3-8及10-12,可特異性辨識酵母菌或細菌所表現之Erns重組蛋白,其抗體亞型皆屬於IgG1,且利用E. coil表現之Erns次單位片段及peptide scanning方法分析此三株單株抗體之抗原辨識位(epitope mapping),結果顯示可分別辨識Erns蛋白胺基酸(amino acid, a.a.)序列161-190、151-170及141-150之位置。進一步選用單株抗體10-12做為捕捉以酵母菌所表現Erns重組蛋白(yErnsN190)為抗原的capture antibody,並建立間接三明治(indirect sandwich)ELISA抗體檢測方法。以經CHEKIT Classical Swine Fever Marker Test (IDEXX) blocking ELISA判定之豬隻血清,包括29支陽性及26支陰性血清進行測定比較,結果顯示本研究所建立之間接三明治ELISA具有良好之敏感性為97% (28/29)而特異性亦可達85% (22/26)。此診斷方法將來可提供作為大量樣品篩檢,亦具有無需純化抗原與操作簡便及價格低廉等優點。
Classical swine fever (CSF) caused by clsaaical swine fever virus (CSFV) is a high mortality disease in pigs. CSFV belongs to the genus Pestivirus of the Flaviviridae family, and infected pigs are often characterized by fever, hemorrhages and leucopenia on pathogenic findings. Glycoprotein Erns of CSFV contains an unique RNase functional domain and could induce humoral immune response, that plays an important role in the pathogenesis. The purpose of this study is to prepare the anti-CSFV Erns monoclonal antibody (Mab) for further applying as a diagnostic tool. Three Mabs 1-41, 3-8 and 10-12 specific to recognize yeast or E. coli expressed Erns were obtained and all were determined to be the type of IgG1 subclass. The antigenic sites recognized by Mabs 1-41, 3-8 and 10-12 were mapped using E. coli subunit and peptide scanning. The result shows to recognize the Erns region of amino acid residues 161-190, 151-170 and 141-150, respectively. Furthermore, the Mab 10-12 was used as a capture antibody to develop a yeast-expressed Erns subunit (yErns/N190) based indirect sandwich ELISA for detecting swine antibody to Erns. The assay demonstrated a high sensitivity of 97% (28/29) and a moderate specificity of 85% (22/26) when compared with a commercial CHEKIT Classical Swine Fever Marker Test (IDEXX) Erns blocking ELISA. This diagnosis tool could detect large numbers serum with easy manipulation and low cost.
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