Liu W, Wang Y T, Tian D S, Yin Z C, Kwang J
Institute of Molecular Agrobiology, The National University of Singapore, Singapore.
Dis Aquat Organ. 2002 Apr 24;49(1):11-8. doi: 10.3354/dao049011.
The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.
从源自马来西亚的感染白斑综合征病毒（WSSV）的虎虾中扩增出编码该病毒包膜蛋白（28 kDa）的vp28基因。重组VP28蛋白（r - 28）在大肠杆菌中表达，并用作制备单克隆抗体（MAb）的抗原。通过基于r - 28抗原的酶联免疫吸附测定（ELISA）筛选出的三种鼠源单克隆抗体（6F6、6H4和9C10）在蛋白质印迹法中也能够识别病毒VP28蛋白以及r - 28。利用这些单克隆抗体在竞争性ELISA中确定了VP28蛋白的三个不重叠表位；因此，借助这些单克隆抗体开发了一种抗原捕获ELISA（Ac - ELISA）。Ac - ELISA能够区分感染WSSV的虾和未感染的虾，并通过聚合酶链反应（PCR）和蛋白质印迹法进一步得到证实。Ac - ELISA能够检测到约400 pg的纯化WSSV样品和20 pg的r - 28，其灵敏度与PCR检测相当，但在检测纯化病毒方面比蛋白质印迹法更灵敏。2000年12月至2001年7月期间从马来西亚一个对虾养殖场采集的血淋巴和组织匀浆样品也通过Ac - ELISA和PCR进行了检测，结果相互印证。
