以酵素連結免疫吸附法偵測雞隻網狀內皮增生病抗體方法之確立

Evaluation of Enzyme-Linked ImmunoSorbent Assays (ELISA) for Detecting Anti- Reticuloendotheliosis Antibody in Chickens

蔡文慈(Wen-Tzu Tsai)

指導教授 ： 王金和

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02607

摘要

多種家禽對網狀內皮增生病毒（Reticuloendotheliosis virus, REV) 具有感受性，其臨床症狀大多輕微主要造成雞隻免疫抑制，生長不良等，少部分會有慢性淋巴腫瘤，其中以runting syndrome對家禽產業造成的經濟損害最大。過去資料顯示台灣地區REV感染情形相當普遍，然而缺乏長期之REV陽性率監測資料。因此本研究之目的為調查台灣土雞REV的盛行率及建立兩種塗鍍重組蛋白質以檢測REV抗體的ELISA。在調查台灣土雞REV的盛行率方面，利用偵測前病毒核酸方式了解台灣土雞REV的陽性率，共計239個屠宰場來源樣本及170個台灣種土雞樣本前病毒核酸偵測結果顯示，屠宰場樣本的REV前病毒場陽性率均高於75％。而三種台灣種土雞品系之REV前病毒檢測結果顯示名古屋系之REV前病毒陽性率（11.1％）低於S系（38.5％）。在兩種檢測抗體的ELISA方面，塗鍍重組蛋白質p30的間接型（indirect) ELISA，與重組封套蛋白質env的阻斷型（blocking) ELISA，以中和試驗結果當作金標準（gold standard)時，計算出此p30 indirect ELISA之敏感性為92% （46/50），特異性為94.8% （91/96）。對照以塗鍍重組蛋白質env做為抗原的阻斷型ELISA敏感性為90% (45/50)特異性為94.8% （91/96）。相較於商品化之ELISA kit敏感性為94% （47/50）、特異性為93.7% （90/96）。由於三種ELISA判讀結果與中和試驗均具有高相關性（p30 indirect ELISA 80.3％, env blocking ELISA 80.1％, I牌商業化套組80.5％)， 故我們推論此兩種塗鍍重組蛋白質以檢測REV抗體之ELISA可做為未來家禽田間調查及快速診斷工具之選擇。

關鍵字

家禽 ； 網狀內皮增生病毒 ； 病毒中和試驗 ； 病毒力價 ； 單株抗體 ； 間接型酵素連結免疫吸附法 ； 間接阻斷型酵素連結免疫吸附法 ； 阻斷型酵素連結免疫吸附法

並列摘要

Naturally occurring avian reticuloendotheliosis virus (REV) infections is common in many domestic fowls. REV causes immunosupression, runting disease, and chronic lymphoma, resulting in a great economic loss. Although REV infection was common, no monitoring data were reported in Taiwan. Thus, the aims of this study were to investigate the REV infection in Taiwan Country chickens and to establish ELISAs for REV antibody detection. For REV monitoring in Taiwan Country chickens, 239 blood samples from a slaughter house and 170 blood samples from a breeder flock were tested for the presence of REV provirus by PCR. The results revealed that REV prevalence in Taiwan Country chicken flocks ranged from 75% to 100% in different kinds of chickens from a slaughterhouse. In the breeder flock, Nogoya breed had a prevalence of 11.1% (4/36) and S breed 38.5% (27/70). Two ELISAs were developed for REV antibody detection. One of the ELISAs was an indirect ELISA coating with recombinant protein p30 and the other was a blocking ELISA with expression env protein. The sensitivity and specificity of p30 ELISA were 92% (46/50) and 94.8% (91/96) and those of blocking ELISA were 90% (45/50) and 94.8% (91/96), respectively. Comparing with a commercial ELISA kit, the three ELISAs were correlated with neutralization result（p30 indirect ELISA 80.3％, env blocking ELISA 80.1％, and a commercial ELISA 80.5％). In conclusion, these two new ELISAs can be applied to detect anti-REV antibody in the field.

並列關鍵字

Avian reticuloendotheliosis virus ； Taiwan Country chicken ； monoclonal antibodies ； serum neutralization test ； indirect ELISA ； indirect blocking ELISA ； blocking ELISA

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