家禽流行性感冒病毒核蛋白抗體之免疫色層分析試片開發

Abstract

檢測抗體為禽流感監測中重要且最常使用之方式，其中包括HI test及ELISA，但由於操作過程繁瑣，需要特殊儀器、試劑，僅適用於實驗室操作，無法應用於現場檢測。因此本研究目的在於建立一快速檢測方式以應用於現場簡便操作。免疫色層分析試片，又被稱為快篩，主要利用固定於硝基纖維膜上的抗原，以及帶有膠體金標示抗體的追蹤子所構成，並能以肉眼快速判讀結果。其中，利用桿狀病毒表現系統表現禽流感病毒之核蛋白，作為固定之抗原，此抗原可與血清中所有亞型之禽流感病毒核蛋白的特異性抗體結合；由於使用重組蛋白，而可降低全病毒於現場操作之風險。以150隻血清進行測試，並以HI test作為金標準，此重組核蛋白之免疫色層分析試片的敏感度為93.2%，特異度為87.5%。此外，以此重組蛋白建立之間接型ELISA測試166隻血清，敏感度為100%，特異度為96.6%。結果顯示本禽流感檢測套組可用於檢測至少H5及H6亞型之AI抗體，並且能在20分鐘內完成判讀，顯示其應用於現場快速檢測之潛力。

Detecting antibody is important and commonly used for AI surveillance, including the HI test and ELISA. However, most of them are time-consuming and require well-equipped laboratories with well-trained medical technicians. For these reasons, they are unsuitable for clinical practice. Hence, to develop a rapid diagnostic kit is the main purpose which can be performed by each person easily. The immunochromatographic strip, also called rapid test, is a membrane-based strip, using gold-labeled antibody as the tracer in order to easily interpret with naked-eye. We used recombinant nucleoprotein as the antigen which can capture the Abs against all AIV strains. The rNP protein was produced by baculovirus expression system which can reduce the risk of virus dissemination when working in the field. The sensitivity and specificity of the rapid strip were 93.2%, and 87.5%, respectively, based on HI test in 150 serum samples. We also developed an indirect NP-ELISA to detect 166 serum samples. The sensitive and specificity were 100% and 96.6%, respecitivelty based on HI test. The results showed this rapid diagnosis strip can detect at least H5 and H6 AI subtypes within 20 minutes, and that is suitable to apply in the field, to achieve quick surveillance purpose.