Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66326
標題: 以三明治酵素連結免疫吸附分析法檢測家禽里奧病毒非結構蛋白σNS抗體
A monoclonal antibody capture enzyme-linked immunosorbent assay for detecting antibodies to avian reovirus nonstructural protein σNS
作者: 陳柏年
Chen, Po-Nien
關鍵字: sandwich ELISA;三明治酵素連結免疫吸附分析法;avian reovirus;家禽里奧病毒
出版社: 獸醫微生物學研究所
摘要: 
中文摘要
以未經His-Bind resin純化之表現蛋白eσNS、經過純化之表現蛋白eσNS、細胞攻毒後之萃取液及病毒液為抗原配合抗家禽里奧病毒非結構蛋白σNS之單源抗體(H1E1)分別建構出以單源抗體捕捉未純化表現蛋白eσNS之三明治ELISA、以單源抗體捕捉細胞攻毒後萃取液之三明治ELISA,另外建構病毒液直接塗鍍之ELISA和純化之表現蛋白eσNS直接塗鍍之ELISA,利用這幾種自製ELISA與商品化ELISA分別檢測SPF雞在不同免疫方式下σNS抗體的變化,並同時進行中和抗體力價測定,結果以單源抗體捕捉未純化表現蛋白eσNS之三明治ELISA,在抗原上所需要的量比直接塗鍍純化過的表現蛋白eσNS所需要的量還要低,且以純化後之eσNS直接塗鍍之ELISA與陰性血清之間的非特異性結合比以單源抗體捕捉未純化表現蛋白eσNS之三明治ELISA來的高。另外,利用單源抗體捕捉未純化表現蛋白eσNS能省去純化表現蛋白eσNS所需花費之時間及成本;所建構之以單源抗體捕捉未純化之表現蛋白eσNS的三明治ELISA能夠區別兩次活毒免疫、一次活毒一次死毒和兩次死毒免疫方式所產生的σNS抗體,而在以單源抗體捕捉細胞攻毒之萃取液三明治ELISA和病毒液直接塗鍍之ELISA對兩次活毒免疫的組別並無法與對照組區分開來。因此,我們以pET28a-σNS之eσNS重組質體進行表現重組蛋白eσNS為抗原配合抗家禽里奧病毒非結構蛋白σNS之單源抗體(H1E1)建構出三明治酵素連結免疫吸附分析法(sandwich ELISA)。以自製之三明治ELISA與商品化之ELISA檢測不同免疫方式下σNS抗體的變化,此三明治ELISA在SPF雞陰性血清的檢測上能降低血清中非特異性結合的情形。將此法應用在檢測四種不同免疫計劃上抗σNS抗體之變化,結果不論一次或兩次死毒免疫注射,其σNS抗體與中和抗體比兩次活毒免疫來的高且持久;在檢測野外血清上,其與中和抗體力價亦成正相關。此三明治酵素連結免疫吸附分析法的建構目的在能夠取代傳統方式,提高特異性、準確性;另外在研究家禽里奧病毒免疫學上亦是一項工具。

Abstract
The antigens without His-Bind resin purified expressed protein(eσNS), with His-Bind resin purified expressed protein, and virion-containing cell extract, virion-containing supernatant combined with monoclonal antibodies(H1E1)to construct sandwich ELISA. At the same time, we also used purified expressed protein and virion-containing supernatant to construct ELISA. We used these self-construct ELISA and conventional ELISA to test the antibodies to σNS from different vaccination schedule, and the serum neutralization. As the result, the amount of antigens used in the sandwich ELISA with non-purified expressed protein were less than indirect ELISA, and ELISA with purified expressed protein had higher non-specific binding reactions than sandwich ELISA. On the other hand, using the sandwich ELISA with non-purified expressed protein could save time and cost on purifying expressed protein. Our self-construct sandwich ELISA with non-purified expressed protein could clearly distinguish the antibodies to σNS from the twice live, one live and one inactive, and twice inactive vaccination. However, the sandwich ELISA with virion-containing cell extract and ELISA with virion-containing supernatant could not distinguish the experiment from the control. Therefore, we use the non-purified antigens eσNS from the expression vector pET28a and combined it with monoclonal antibodies to construct sandwich ELISA. This sandwich ELISA not only could decrease the non-specified binding reactions but also had high relation with the result of serum neutralization test. The purpose to construct the sandwich ELISA was to find a more specific and efficient tool. By this tool, we could study the immunological concept of avian reovirus.
URI: http://hdl.handle.net/11455/66326
Appears in Collections:微生物暨公共衛生學研究所

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