A型禽流感病毒核蛋白單源抗體於免疫化學染色之應用

2013-11-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/672206摘要：家禽流行性感冒病毒 (Avian influenza virus, AIV) 除了造成養禽場重大經濟損失外，因其具人畜共通之風險，近年來為世界所關注，快速、敏感性及特異性高的診斷方法或檢測工具便有迫切需求，而單源抗體 (MAb) 憑著對抗原的高特異性及專一性，乃為生醫領域所廣泛應用。本研究室利用2003 年台灣分離到的病毒株A/chicken/Taiwan/1209/03 (H5N2)，製備出抗AIV 核蛋白(nucleoprotein, NP)的MAb WF-4，經西方墨點法 (Western blot assay, WB) 顯示其可辨識數種不同亞型流感病毒與 NP 分子量 56 kDa 相同的病毒抗原，及真核表現 AIV NP 基因重組 (recombinant) 產物，總結其辨識抗原特異性為 NP。為測試 WF-4 用於病毒診斷之能力，實驗感染 H5N2 病毒雞所採集病理檢測生材以 WF-4 為免疫化學染色法(immunohistochemistry, IHC)之一抗，於具病毒親合性之組織檢體皆可測到陽性反應；再以十五種 HA 亞型流感病毒中的十種，感染狗腎細胞(Madin-Darby canine kidney, MDCK)後的中性福馬林固定樣品，以 WF-4 為一抗的免疫螢光染色(immunofluorescence assay, IFA)測試結果皆為陽性，邏輯推論WF-4 可適用於 A 型流感病毒的病理 IHC 檢測(及 WB 實驗室檢測)，本計畫擬完成十五種亞型 AIV 病理檢體WF-4 MAb 之IHC 測試結果，其純化製程，及純化製品應用於 IHC 之最佳使用條件，如抗體稀釋濃度等具體流程及最優化(optimization) 條件，完全確認，並轉予合作廠商之技術人員，使熟習操作，俾為後續開發成IHC 及 WB 檢測 A 型流感病毒商品之製程基礎。<br> Abstract: Avian influenza virus (AIV), a devastating viral disease of poultry which causes high impact on either animal or human health, has become a matter of increasing public concern and scientific interest within the last decade. The accurate and prompt diagnosis of AIV infection in birds is a critical component of a disease control plan. Using of monoclonal antibodies (MAbs) increases the specificity, accuracy and efficiency of diagnosis compared to polyclonal antibodies and provides an unlimited quantity and consistent quality of reagents. In 2003, an AIV of H5N2 subtype A/chicken/Taiwan/1209/03 (H5N2/1209) was isolated from Taiwan. MAb WF-4 anti-H5N2/1209, the first H5N2 isolate in Taiwan, was obtained in our laboratory lately. Western blotting analysis with baculovirus expressed recombinant nucleoprotein (rNP) indicated that WF-4 recognized AIV NP. The application of WF-4 on the pathological diagnosis was confirmed firstly by immunohistochemistry (IHC) staining on H5N2/1209 experimentally infected chicken and by immunofluorescence assay (IFA) staining on ten different serotypes of AIV infected susceptible cell line MDCK (Madin-Darby canine kidney) fixed by neutral formalin. Therefore, WF-4 would be full-filled the characteristic need of type A influenza virus by IHC in diagnostic (and WB in research) laboratory. This project intends to complete the final test of applying WF-4 on IHC pathological diagnosis of all serotypes type A AIV. Also, the standard operation procedures of MAb purification, optimal dilution of MAb for IHC (and WB) will be established and transferred to the cooperative partner during the project year for subsequent development of market product.家禽&#63946&#64008性感冒病毒核蛋白免疫化學染色法Avian influenza virusnucleoproteinimmunohistochemistryA型禽流感病毒核蛋白單源抗體於免疫化學染色之應用