Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/12671
標題: 建立以競爭性酵素連結免疫吸附分析法快速診斷H5亞型高病原性家禽流行性感冒之技術
Establishment of competitive enzyme-linked immunosorbent assay for Rapid diagnosis of highly pathogenic avian influenza virus of H5 subtype
作者: 邱文鴻
Chiu, Wen-hung
關鍵字: Avian influenza virus
家禽流行性感冒
competitive enzyme-linked immunosorbent assay
競爭性酵素連結免疫吸附分析
出版社: 獸醫學系
摘要: 家禽流行性感冒(Avian Influenza, AI)是由A型流行性感冒病毒所引起之全身性或呼吸性感染的疾病。依其表面醣蛋白抗原血球凝集素(Hemagglutinin, HA)可分為15種血清亞型(H1~H15)、而依神經胺酸酶(Neuraminidase, NA)亦可分為9種血清亞型(N1~N9)。其中H5及H7可能為強毒型AI病毒,感染後可能會造成嚴重的全身症狀及高死亡率。傳統上區分AI病毒血清亞型的方法為血球凝集抑制試驗(hemagglutination inhibition test, HI),但此方法缺乏敏感性且耗時。本研究利用H5血清亞型之HA重組蛋白,經E. coli BL21(DE3)表現後純化蛋白免疫BALA/c小鼠,製備出H5血清亞型之HA單源抗體,單源抗體經純化後進行競爭性ELISA (competitive enzyme-linked immunosorbent assay, CELISA)之試驗,以期能建立強毒型AI病毒H5血清亞型之快速診斷技術。實驗結果顯示H5血清亞型之單源抗體僅能被H5血清亞型之高免血清所競爭。且有極高的特異性。H5高免血清在512HI抗體力價與單源抗體10號競爭結果有91.5的競爭百分比。而與單源抗體11號的試驗結果,有86.1的競爭百分比。因此利用原核系統表現之H5血清亞型HA重組蛋白,配合以H5血清亞型重組蛋白製備之單源抗體所建立之競爭性ELISA套組,可用於偵測H5強毒型AI之抗血清,此一診斷技術的建立將可協助高病原性H5血清亞型AI病毒之診斷。
Avian Influenza (AI) is caused by type A influenza virus, which might cause a systemic or respiratory infection and disease. According to their surface glycoprotein, fifteen HA subtypes (HA1-HA15) and nine NA subtypes (NA1-NA9) have been identified. Avian influenza viruses of subtypes H5 and H7 might be highly pathogenic, which cause a severe systemic disease and a high mortality after infection. The conventional method for differentiating HA subtypes of AIV is hemagglutination inhibition test (HI test), which is both time-consuming and insensitive. In this study, we used E. coli BL21 (DE3) as the host to express recombinant HA protein of H5 subtype, and immunized BALB/c mice with the recombinant HA protein to prepare monoclonal antibody against the HA protein. After purification, the recombinant HA protein and the monoclonal antibody were used to develop a competitive-ELISA that could differentiate the antisera of H5 subtype from antisera other subtypes. Our results show that, for this competitive-ELISA, the binding of H5 monoclonal antibodies could be specifically competed by H5-specific serum. The H5-specific serum (512 HI), showed 91.5% and 86.1% of competition when competed with monoclonal antibody number 10 and number 11. Therefore, the competitive-ELISA developed by using recombinant HA proteins and monoclonal antibody, might serve as a tool for rapid identification of highly pathogenic influenza virus of the H5 subtype.
URI: http://hdl.handle.net/11455/12671
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