請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49231
標題: | 以抗Site 2單源抗體與口蹄疫類病毒空殼蛋白建構阻斷型ELISA對免疫動物進行血清學監控 Development of a blocking ELISA based on site 2 monoclonal antibodies and foot-and-mouth disease virus-like particles for seromonitoring vaccinated animals |
作者: | Heng-Wei Lee 李恆瑋 |
指導教授: | 鄭益謙(Ivan-Chen Cheng) |
關鍵字: | 口蹄疫,單源抗體,Site 2中和抗原決定位,類病毒空殼蛋白,阻斷型酵素連結免疫吸附試驗, foot-and-mouth disease,monoclonal antibodies,antigenic site 2,virus-like particles,blocking enzyme-linked immunosorbent assay, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 口蹄疫可感染所有偶蹄類動物,且具有高度傳染力,在畜牧產業中是相當重要的病毒性疾病。目前,台灣的防疫政策仍然是全面施打不活化疫苗,並以血清中和試驗(serum neutralizing test, SN test)來評估免疫動物獲得中和抗體保護力的情形。然而,SN test耗時、成本高,且需在嚴格的負壓實驗室內操作。近期的研究指出,大多免疫動物的血清中以抗Site 2抗體為主。因此,我們希望以類病毒空殼蛋白(virus-like particles, VLP)和抗Site 2單源抗體(monoclonal antibody, MAb)建構阻斷型酵素連結免疫吸附試驗(blocking enzyme-linked immunosorbent assay, bELISA),並評估是否可取代SN test。VLP的製備以哺乳類細胞表現系統-transient expression assay搭配共轉染策略製造,並經過蔗醣梯度離心(sucrose gradient centrifugation)及sandwich ELISA等實驗間接確定VLP構型。為了從本實驗室已製備的41株MAbs中挑選出抗Site 2抗體,本研究採用單點突變(knock-out mutagenesis)的方式,以免疫螢光染色(immunofluorescence assay, IFA)和indirect ELISA找出可以辨識VLP但無法辨識Site 2單點突變VLP(mutated VLP, mVLP)之MAbs。配合中和抗體力價檢測結果,有6株抗Site 2抗體被篩選出來。經過sandwich ELISA以及少量樣品測試(實驗豬隻血清和臨床檢體)之bELISA,挑選S11E-9為最佳tracer完成bELISA的建構。 Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals worldwide. Serum neutralization test (SN test), a gold standard for evaluating the protection rate against FMDV, is still performed for disease control in Taiwan. However, SN test is laborious, expensive and requires a high-containment biosafety lab. Based on the current study on vaccinated animals, antigenic site 2 of FMDV is the most immuno-dominant neutralizing site. We aim to establish a blocking ELISA (bELISA) based on FMD virus-like particles (VLPs) and site 2 monoclonal antibody (MAb) to detect antibodies against site 2 from vaccinated animals and replace the SN test. VLPs were expressed by eukaryotic transient expression assay with co-transfect strategy and examined by sucrose gradient centrifugation accompanied with sandwich ELISA. For mapping MAb against site 2 from 41 anti-FMDV MAbs prepared previously, we performed knock-out mutagenesis with VLPs and mVLPs (site 2 mutated) by immunofluorescence assay (IFA) and indirect ELISA. Combined with neutralization assay, results indicated that 6 MAbs recognized site 2. Based on the results of sandwich ELISA and bELISA with experimental serum, S11E-9 was regarded as the best tracer to establish bELISA with VLPs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49231 |
DOI: | 10.6342/NTU201603059 |
全文授權: | 有償授權 |
顯示於系所單位: | 獸醫學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 6.7 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。