Zhang Xuemei, Xu Jingwen, Wu Zhongxiang, Zhu Wenbing, Dong Shaozhong
a Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases , Kunming , Yunnan , China.
J Immunoassay Immunochem. 2018;39(2):136-149. doi: 10.1080/15321819.2017.1344128. Epub 2018 Jun 22.
Tree shrews (Tupaia belangeri) are small squirrel-like mammals closely related to primates. Due to their susceptibility to several human viruses, tree shrews have been proposed as potential animal models for the study of human viral infections. However, there are no standardized assays currently available for the detection of tree shrew-specific interferon (IFN)-γ, a major cytokine secreted during the antiviral immune response. Herein, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the quantification of IFN-γ in tree shrew serum samples. Tree shrew-specific IFN-γ was expressed in Escherichia coli via fusion with glutathione S-transferase (GST-TS-IFN-γ) to obtain recombinant IFN-γ. To generate anti-IFN-γ monoclonal antibodies, mice were immunized with the GST-TS-IFN-γ recombinant fusion protein, and hybridoma cell lines were established. Similarly, anti-IFN-γ polyclonal antibodies were obtained from immunized rabbits, purified, and conjugated to horseradish peroxidase (HRP). Based on the results obtained from the antibody matching test, we optimized the monoclonal antibody (1:2000) and the HRP-conjugated polyclonal antibody (1:8000) as coating and detection antibodies, respectively. Titration curves were generated with recombinant IFN-γ to develop a sensitive sandwich ELISA; the lowest detection limit of the assay was 20 ng/mL. We also tested mitogen-stimulated tree shrew blood samples in this ELISA, and found significantly higher levels of IFN-γ in the stimulated versus the unstimulated samples. Most importantly, our ELISA system detected native IFN-γ in serum samples from 50 healthy tree shrews. We have thus developed a novel ELISA, and have demonstrated the first ELISA-based measurement of IFN-γ in tree shrew serum samples.
树鼩（Tupaia belangeri）是一种与灵长类密切相关的小型松鼠状哺乳动物。由于它们对多种人类病毒易感，树鼩已被提议作为研究人类病毒感染的潜在动物模型。然而，目前尚无用于检测树鼩特异性干扰素（IFN）-γ的标准化检测方法，IFN-γ是抗病毒免疫反应期间分泌的一种主要细胞因子。在此，我们开发了一种新型酶联免疫吸附测定（ELISA）方法，用于定量树鼩血清样本中的IFN-γ。树鼩特异性IFN-γ通过与谷胱甘肽S-转移酶（GST-TS-IFN-γ）融合在大肠杆菌中表达，以获得重组IFN-γ。为了产生抗IFN-γ单克隆抗体，用GST-TS-IFN-γ重组融合蛋白免疫小鼠，并建立杂交瘤细胞系。同样，从免疫兔中获得抗IFN-γ多克隆抗体，进行纯化，并与辣根过氧化物酶（HRP）偶联。根据抗体匹配试验的结果，我们分别优化了单克隆抗体（1:2000）和HRP偶联多克隆抗体（1:8000）作为包被抗体和检测抗体。用重组IFN-γ生成滴定曲线，以开发一种灵敏的夹心ELISA；该检测方法的最低检测限为20 ng/mL。我们还在该ELISA中检测了丝裂原刺激的树鼩血液样本，发现刺激样本中的IFN-γ水平明显高于未刺激样本。最重要的是，我们的ELISA系统检测了50只健康树鼩血清样本中的天然IFN-γ。因此，我们开发了一种新型ELISA，并首次证明了基于ELISA方法对树鼩血清样本中IFN-γ的测量。
