應用奈米金粒子之於豬瘟病毒E2抗原之檢測

Abstract

典型豬瘟(CSF)是一種豬瘟病毒所引起的高度致命的豬傳染性疾病，導致許多國家養豬業的大量經濟損失。因此，如何開發簡單、快速、有效診斷典型豬瘟病毒的分析方法極為重要，以減少養豬業所受的損害與衝擊。當豬隻被感染後，會大量產生抗體對抗豬瘟病毒的E2醣蛋白，因而E2醣蛋白被視為診斷典型豬瘟的關鍵生物標的。因此，我們開發了一系列利用抗體(Ab)、山葵過氧化酶 (HRP)與奈米金粒子鍵結作為偵測平台(Ab/HRP-AuNPs)的方法，例如酵素連結免疫吸收試驗(ELISA)、快速檢測試片、電流式免疫感測器來檢測豬瘟病毒E2抗原。在直接型ELISA和三明治型ELISA實驗中，Ab/HRP-AuNPs對於E2抗原展現良好的靈敏度。直接型ELISA的線性偵測範圍從1.95±0.10到31.25±2.05 fmoles, 而三明治型ELISA 則是從125±13.30到4000±583.43 fmoles。至於偵測極限，直接型ELISA和三明治型ELISA分別是1.95±0.10及61.25±5.36 fmoles。關於免疫快速診斷試片的開發，對於E2抗原的偵測範圍從2.25到22 pmoles，偵測極限達2.25 pmoles。

Classical swine fever (CSF) is a highly contagious and fatal disease of swine due to the infection of CSF virus (CSFV), causing considerable economic loss in swine industry of many countries. Therefore, the development of the simple, quick and effective analytic methods for the detection of CSFV infection is essential in reducing the damage of swine industry. The antibody specifically against glycoprotein E2 of CSFV (E2 antigen) was found to be largely booted in the infected pigs. Accordingly, E2 antigen was identified to be the key biomarker for the diagnosis of CSF. Therefore, various detection methods for the detection of E2 antigen based on the antibody and horseradish peroxidase (HRP) conjugated gold nanoparticles (Ab/HRP-AuNPs), including ELISA and rapid test strip, were design, developed and characterized in this study. The Ab/HRP-AuNPs exhibited a high sensitivity toward E2 antigen on the direct or sandwich type ELISA assay format. The linear detection range is 1.95±0.10 to 31.25±2.05 fmoles with direct ELISA and 125±13.30 to 4000±583.43 fmoles with sandwich ELISA. The detection limit was 1.95±0.10 fmoles and 62.5±5.36 fmoles for direct and sandwich ELISA, respectively. The flow assay strip was developed for a quick detection of E2 antigen exhibiting a detection range from 2.25 to 22 pmoles with a detection limit of 2.25 pmoles.