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標題: 大腸桿菌表現之傳染性華氏囊病病毒VP243蛋白應用於四種ELISA之研發
Development of four types of ELISAs coating E. coli expressed VP243 for detecting antibody against infectious bursal disease virus
作者: 顏竹君
Yen, Chu-Chun
關鍵字: 傳染性華氏囊病病毒;IBDV;酵素連結免疫吸附分析法;ELISA
出版社: 微生物暨公共衛生學研究所
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傳染性華氏囊病病毒 (Infection bursal disease virus;IBDV) 具有高度傳染性且造成年幼雞隻免疫力下降,而目前常以酵素連結免疫吸附分析法 (enzyme linked immunosorbant assay; ELISA) 方式來診斷此疾病。傳統上以IBDV全病毒ELISA來檢測,因先前研究指出重組VP243可形成類病毒顆粒 (virus like particle; VLP),故本實驗以原核系統E. coli表現重組的IBDV多蛋白VP243當作抗原應用於ELISA之檢測上。實驗檢測206隻野外雞隻血清,並以商品化之IDEXX IBD ELISA檢測結果當作黃金標準。將自製VP243-ELISA分為間接型 (Indirect) ELISA、三明治型 (Sandwich) ELISA、阻斷型 (Blocking) ELISA,其中間接型ELISA依VP243純化方式不同分為以Ni-NTA純化及以氯化銫 (CsCl) 密度梯度離心純化;而三明治型、阻斷型ELISA則以未純化的VP243可溶蛋白當作抗原,並配合市售的Anti-VP2單株抗體之應用,此四種ELISA分別簡稱為Ni-NTA VP243-ELISA、CsCl VP243-ELISA、VP243 Sandwich ELISA、VP243 Blocking ELISA。本實驗以兩種裁切點判定方式,傳統上非同源陰性血清平均值加上標準差當作截切點,和以Two-graph receiver operating characteristic (TG-ROC) 選擇之截切點,並比較其ELISA之結果。結果顯示Ni-NTA VP243-ELISA以血清平均值加上標準差當做截切點時,當加上12個標準差時其敏感性、特異性及準確率分別為99.2%、93.83%及97.09%,而當以TG-ROC判定截切點時其敏感性、特異性及準確率分別為97.6%、95.06%及96.6%,且ROC曲線下面積 (area under the ROC curve; AUC) 達0.99表示其為判別力良好的檢測方式,而且Ni-NTA VP243-ELISA具有比其他三種ELISA較高之敏感性、特異性、準確率。整體而言Ni-NTA VP243-ELISA是有效檢測方式且成本低廉操作簡便,可應用於未來檢測IBDV的診斷。

Infection bursal disease virus (IBDV) causes a highly contagious disease and induces immunosuppression in young chickens. The enzyme-linked immunesorbent assay (ELISA)was commonly used in the field to evaluate the efficiency of IBD vaccination and monitor the infection of IBDV. The commercial IBD ELISA was based on the use of whole virus as the antigens. The researches have demonstrated that the recombinant VP243 could form virus-like particles (VLPs), therefore, the objective of this study was to develop different types of ELISAs using E. coli expressed VP243 as coating antigen for detection of antibody against IBDV. In-house VP243-ELISAs including two indirect ELISAs, sandwich ELISA and blocking ELISA were compared with commercial IDEXX IBD ELISA. The indirect ELISA was coated with the Ni-NTA-purified VP243 or CsCl purified VP243. The sandwich ELISA was coated with the commercial anti-VP2 monoclonal antibody to capture the soluble VP243 from the E. coli lysate. The blocking ELISA was coated with the soluble VP243 from the E. coli lysate and detected by the commercial anti-VP2 monoclonal antibody. These four types of VP243-ELISA were in short for Ni-NTA VP243-ELISA, CsCl VP243-ELISA, VP243 sandwich ELISA and VP243 Blocking ELISA. Total 206 sera from field were used for the evaluation and the commercial IDEXX IBD ELISA coated with whole virus was used as gold standard method. The cut-off values were determined by mean plus standard deviations or the two-graph receiver operation characteristics (TG-ROC). When the Ni-NTA VP243-ELISA cut-off value was set on that mean plus 12 standard deviations, the sensitivity, specificity and accuracy were 99.2%, 93.83% and 97.09%, respectively. When the cut-off value was determined by TG-ROC, the sensitivity, specificity and accuracy were 97.6%, 95.06% and 96.6%, respectively. Moreover, the area under the ROC curve (AUC) of the Ni-NTA VP243-ELISA was 0.99, indicating that this test is a highly efficient method. The results showed that Ni-NTA VP243-ELISA had higher sensitivity, specificity and accuracy than those in other VP243-ELISAs. Therefore, Ni-NTA VP243-ELISA was demonstrated to be an effective and economical ELISA for detection of IBDV infection.
其他識別: U0005-1908201200282300
Appears in Collections:微生物暨公共衛生學研究所

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