Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/93092
標題: The Development of ELISA for the Detection of the Antibody against Porcine Reproductive and Respiratory Syndrome Virus
豬生殖與呼吸綜合症抗體檢測試劑之研發與應用
作者: 黃儀婷
Yi-Ting Huang
關鍵字: 豬生殖與呼吸綜合症
Porcine Reproductive and Respiratory Syndrome
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摘要: Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease of domestic swine worldwide caused by PRRS virus (PRRSV). The typical clinical signs of PRRS are severe reproductive failure in sows and respiratory problems in pigs of all ages. Currently, ELISA is usually used for the detection of antibody against PRRSV. The advantage of ELISA are time-efficient and cost-effective. The objective of this study was to develop the ELISAs coated with various PRRSV proteins for the detection of antibody against PRRSV. In-house ORF7 ELISA (coated with 15 KDa N protein), Nsp2 ELISA (coated with partial Nsp2 protein), and 14-mer ORF7 ELISA (coated with 14-mer N protein) were used to detect anti-PRRSV antibody. The N protein and Nsp2 protein were expressed by E.coli and purified by affinity chromatography and the 14-mer ORF7 was a 14-mer synthesized peptide. Total 34 pig sera were used for the evaluation and the commercial IDEXX PRRS ELISA was used as gold standard method. The cut-off values were determined by mean plus several standard deviation or the two-graph receiver operation characteristics (TG-ROC) and the TG-ROC gave better results for all three ELISAs. According to the TG-ROC method, the sensitivity, specificity and accuracy were (100 %, 94.10 %, 96.96 %), (91.67 %, 100 %, 96.5 %) and (93.75 %, 88.24 %, 90.9 %) for ORF7 ELISA, Nsp2 ELISA, and 14-mer ORF7 ELISA, respectively. The results showed that ORF7 ELISA had higher sensitivity, specificity and accuracy than those of other in-house ELISAs. ORF7 ELISA is suitable for the detection of anti-PRRSV antibody.
豬生殖與呼吸綜合症 (porcine reproductive and respiratory syndrome; PRRS) 由豬生殖與呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus; PRRSV)所引起,主要病徵為母豬的繁殖障礙,及各年齡層豬隻的呼吸道疾病,引起豬隻產業嚴重經濟損失,因此豬場中監控PRRS的情況就變得重要。酵素連結免疫吸附分析法 (enzyme linked immunosorbant assay; ELISA)是目前最常使用的檢測方法之一,其優點不僅省時省成本,並可一次檢測大量檢體。本實驗之主旨為利用PRRSV的不同蛋白質做為抗原並應用於ELISA上,用來檢測豬隻血清中抗PRRSV的抗體。本實驗共有3種ELISA,分別為ORF7 ELISA、Nsp2 ELISA、14-mer ORF7 ELISA,ORF7 ELISA、Nsp2 ELISA中之抗原為構築之重組質體並以大腸桿菌表現,經親和層析純化之ORF7或部分Nsp2蛋白;而14-mer ORF7 ELISA之抗原則是選取PRRSV兩種基因型在N蛋白之相異片段並以此合成之胜肽。本實驗共有34個豬隻血清進行檢測,並以商品化的IDEXX PRRS ELISA的檢測結果視為黃金標準,再將全數血清以自製ELISA檢測之,並以IDEXX PRRS ELISA檢測之陰性血清,在自製ELISA中檢測的讀值平均值及標準差,推算截切點;也用Receiver operating characteristic (ROC)曲線及Two-graph -ROC (TG-ROC)判定截切點,再比較兩種方式的截切點及結果。由結果顯示ORF7 ELISA以平均值加上兩倍標準差時,其敏感性、特異性、準確度分別為93.75 %、94.11 %、93.94 %;以TG-ROC判定最佳截切點時其敏感性、特異性、準確度分別為100 %、94.10 %、96.96 %,而ROC曲線下面積為0.99,代表具有良好的判別力,並且ORF7 ELISA結果與IDEXX PRRS ELISA結果的相關係數分析為0.76,由這些結果可知ORF7 ELISA與其他兩種ELISA相比有較高的敏感性、特異性、準確度,經實驗證明ORF7 ELISA為成本低廉且操作方便的檢測方法,並可應用於未來PRRS的檢測。
URI: http://hdl.handle.net/11455/93092
文章公開時間: 2018-08-14
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