Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/93090
標題: Characterization and Application of the Monoclonal Antibody against Porcine Circovirus Type 2 Capsid Protein
豬環狀病毒第二型Cap蛋白之單株抗體的分析及應用
作者: 張瑜芝
Yu Chih Chang
關鍵字: 豬環狀病毒第二型
外殼蛋白
單株抗體
Porcine Circovirus Type 2
Capsid Protein
Monoclonal Antibody
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摘要: Porcine circovirus type 2 (PCV2) is recognized as a primary cause in swine post-weaning multisystemic wasting syndrome (PMWS). The PCV2 open reading frame 2 (ORF2) gene expresses capsid protein (Cap) which is the sole viral structural protein containing major antigenic epitopes. The purpose of this study is to prepare the monoclonal antibody (MAb) against the PCV2 Cap for further developing as diagnostic reagents. A monoclonal antibody, G9/E2, was obtained by immunization of BALB/c mice with the Ingelvac® CircoFLEX commercial vaccine followed by the hybridoma experiment. The MAb G9/E2 with the IgG1 subclass could specifically recognize the PCV2-infected PK-15 cell by indirect immunofluorescence assay but failed to react with the denatured Cap protein in the epitope mapping analysis. In addition, the MAb G9/E2 could be applied to detect PCV2-infected swine tonsil tissue in immunohistochemistry (IHC) test. Furthermore, the MAb G9/E2 was conjugated with the peroxidase and used as a detecting antibody in company with another previously made MAb D9 as a capturing antibody for further developing a quantitative direct sandwich ELISA. The results demonstrated effective quantitation of Cap protein at the concentration 0.22-3.67 μg/ml and 0.195-12.5 μg/ml when using Ingelvac® CircoFLEX and E. coli expressed Cap as strand, respectively. The coefficient of determination (R2) could reach 0.97 and 0.98, respectively.
豬環狀病毒第二型(porcine circovirus type 2, PCV2)是引起離乳後豬隻多系統性消耗症候群(post-weaning multisystemic wasting syndrome, PMWS)之主要病因。病毒其第二開讀框基因(ORF2)轉譯出的病毒外殼蛋白(capsid protein, Cap protein)為唯一的結構蛋白並包含重要之抗原決定位。本研究之目的為製備抗Cap蛋白之單株抗體,並進一步開發做為診斷試劑。首先利用商業化之 Ingelvac® CircoFLEX疫苗製備疫苗抗原並進行小鼠免疫,經由融合瘤試驗進而得到一株單株抗體命名為G9/E2,以間接螢光抗體分析結果顯示能特異性辨識PCV2感染之細胞。此單株抗體之亞型為IgG1,且經抗原決定位分析結果顯示為辨認構形依賴型抗原決定位。而與豬隻免疫血清進行blocking ELISA試驗之結果顯示不具有競爭性。進一步應用於免疫組織化學染色法(immunohistochemistry, IHC)分析,顯示G9/E2抗體能特異性辨識PCV2感染之扁桃腺組織。另外,將單株抗體G9/E2連結過氧化酶(peroxidase)製成conjugate做為偵測抗體,配合另一株單株抗體D9做為捕捉抗體可建立直接型三明治ELISA抗原定量檢測方法。結果顯示,以疫苗抗原做為標準品時可定量抗原濃度範圍為0.013至0.91 μg/ml,而以大腸桿菌表現之Cap重組蛋白做為標準品時可定量範圍介於0.39至25 μg/ml,而且決定係數(coefficient of determination, R2)則分別為0.97及0.98。
URI: http://hdl.handle.net/11455/93090
文章公開時間: 2018-06-22
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