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標題: 鴨肝炎病毒一型VP1蛋白酵素連結免疫吸附分析(ELISA)套組開發與單源抗體製備
Development of ELISA Kit and Monoclonal Antibody for Duck Hepatitis A Virus VP1 Protein
作者: 王韻婷
Wang, Yun-Ting
關鍵字: Duck Hepatitis A Virus
Monoclonal Antibody
出版社: 獸醫學系暨研究所
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摘要: 鴨肝炎病毒一型(DHAV-1)是一種在雛鴨高度致死性疾病,台灣曾經在1972年及1990年分別爆發大流行。在本研究中先利用血清中和試驗調查2010年台灣的一日齡雛鴨與待售成鴨血清中DHAV-1中和抗體力價,其抗體陽性率分別為18.78 %及10.5 %,證明在台灣鴨場環境中仍有DHAV-1野外毒株持續潛伏。因此利用鴨肝炎病毒一型野外分離株之VP1蛋白進行DHAV-1 抗體ELISA檢測系統的建構,期望可以取代傳統的血清中和試驗,減少檢測所需耗費的時間。首先使用原核E.coli表現系統表現並純化VP1蛋白,再以DHAV-1高免血清及陰性鴨隻血清使用於西方墨點法來確認VP1蛋白的特異性。相對於血清中和試驗,使用純化VP1蛋白建構的ELISA之相對敏感性為與相對特異性分別為100 %與94.64 %,一致性則為0.93。顯示將純化VP1蛋白使用於ELISA與血清中和試驗一樣具有良好的特異性,且需要耗費的時間較短,所以使用VP1蛋白建立ELISA檢測套組可達到快速且大量檢測抗體樣本的目的。進一步實驗利用所純化的DHAV-1重組蛋白VP1免疫BALB/C小鼠,來製備出針對DHAV-1 VP1蛋白的單源抗體,未來可將其應用於檢測套組開發。經過細胞融合、多次篩選和單株化後共篩選出1株具有特異性分泌抗VP1蛋白抗體能力且敏感性佳的單株抗體,以dot-enzyme-linked immunosorbent assay評估所製備的單源抗體2C2有良好的敏感性,由間接免疫螢光染色法與免疫過氧化酶單層細胞分析法證明單源抗體2C2具有良好特異性。因此本實驗所製備之單源抗體未來可應用於開發診斷試劑,有利於DHAV-1的監控。
Duck hepatitis A virus type I is a highly fatal disease of young duckling. In Taiwan two epidemics of duck viral hepatitis occurred in 1972 and 1990. Therefore, development of a fast and sensitive method to detect DHAV-1 is essential to control the disease. In this study, serum neutralization test was used to investigate the neutralize antibody titer for adult duck and one day duck. The positive rate of antibody titer is 18.7 % and 10.5 %. It was proved wild-type DHAV-1 was still in natural environment. A simple and reliable ELISA for detecting DHAV-1 specific antibodies using DHAV-1 VP1 protein expressed from E. coli as a coating antigen was developed in order to replace traditional antibody determination. The sensitivity and specificity of the ELISA were evaluated in comparison with serum neutralization tests. The sensitivity and specificity were found to be 100% and 94.46 %, respectively. The kappa value between the two methods was 0.93. The results suggest that the VP1-based ELISA has advantages over conventional serum neutralization tests and may be applied for detection of a larger number of samples. In order to further increase sensitivity and specificity for detecting DHAV-1, we attempted to develop VP1 protein-based monoclonal antibody named 2C2. Thus far, the synthesized VP1 monoclonal antibody has both sensitivity and specificity against VP1 protein as determined by dot-enzyme-linked immunosorbent, IFA and IPMA. Taken together, our current results suggest that DHAV-1 VP1 protein-based monoclonal antibody has potential to develop new detection tools for DHAV-1 surveillance.
其他識別: U0005-2706201113475600
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