家禽網狀內皮增生症病毒酵素連結免疫吸附法診斷試劑之開發

Abstract

家禽網狀內皮增生症病毒(Avain reticuloendotheliosis virus, REV)可自然感染多種鳥禽類，包括台灣主要的經濟動物雞、鴨與鵝，雖然被感染的家禽於臨床上並未出現明顯的病徵，卻會引起飼養戶很大的經濟損失。目前網狀內皮增生症(reticuloendotheliosis)於現場尚未有有效預防及控制的方法，因此，本研究之目的為開發檢測REV抗原及抗體之酵素連結免疫吸附法(ELISA)，將此經濟、快速、準確的檢測方式應用於快速診斷；並針對台灣水禽病例嘗試分離REV，藉由序列分析推估其親源關係；同時亦對本實驗室現有的三株抗體進行抗原決定位分析(epitope mapping)，評估三株單株抗體辨識REV之能力。本研究共建立三種ELISA：以抗REV單株抗體及多株抗體分別作為捕捉抗體及偵測抗體，開發間接型抗原捕捉ELISA (iAC-ELISA)來偵測檢體中之REV抗原；此外，分別以原核系統表現REV之封套蛋白及p30蛋白作為塗鍍抗原，建立阻斷型ELISA及間接型ELISA來偵測血清中的抗REV抗體。本研究總共自水禽分離到4株REV，這些分離株無論是env或LTR序列以親源樹圖分析皆屬於同一群，此外我們也在鵝隻的腫瘤組織中偵測到與部份v-rel基因相似的序列；在抗原決定位分析方面，三株單株抗體所辨識的區域皆是屬於REV封套蛋白基因的高保留序列區；在偵測REV抗原方面，iAC-ELISA確實可以辨別病毒液中之REV；同為檢測REV抗體之阻斷型ELISA與間接型ELISA，兩者與商品化ELISA kit所判讀之結果相關性皆很高(98.7及96.7％)。因此，可利用本研究所建立之ELISA檢測REV抗原及抗體，做為田間調查及快速診斷之有效工具。

Nature hosts of avian reticuloendotheliosis virus (REV) infection include chickens, ducks and geese, which are the main commercial poultry in Taiwan. Without treatment and control procedures for reticuloendotheliosis, the effect of impair immune responses against many pathogens by REV can procure severe economic losses in commercial poultry. The purpose of this study was to develop rapid, convenient and economic methods for detecting REV antigen and antibody. Virus isolation, sequencing, and phylogenetic analysis were performed for goose and duck REVs. The epitopes that the three monoclonal antibodies (mAbs) identified were also determined by using three different prokaryotic expression env proteins. For antigen detection, an indirect antigen capture enzyme-linked immunosorbent assay (iAC-ELISA) was developed with a mAb as the capture antibody and a polyclonal antibody as the detector antibody. For antibody detection, a prokaryotic expression env protein and p30 protein were coated in a blocking enzyme-linked immunosorbent assay (ELISA) and an indirect ELISA, respectively. After 2 passages in DF1 cell, four REVs were isolated from geese and ducks and their env and long terminal repeat sequences were closely related to each other. In addition, a partial v-rel-like sequence was detected in a goose tumor. All the three mAbs could identify the env conserved region of REVs from chickens and geese. For antigen detection, REVs from chickens, geese and ducks could be identified by the iAC-ELISA. For Antibody detection, both the blocking ELISA and the indirect ELISA were highly relative to the commercial ELISA kit with 98.7% and 96.7% agreements, respectively. In conclusion, these methods could be used for anti-REV antibody and antigen detections in the field.