Scientific Research Department, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.
Scientific Research Department, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.
J Immunol Methods. 2018 Oct;461:63-72. doi: 10.1016/j.jim.2018.06.007. Epub 2018 Jun 9.
Cell lines stably transfected with genes responding to Type I interferons (IFN) are potentially a useful alternative to enzyme linked immuo-assays (ELISAs) or assays based on resistance of a test cell line to virus infection using cell death or infection endpoints. Increasingly available are a variety of commercial cell lines developed for reporter gene assays (RGAs) which are responsive to IFN exposure. These cells produce a soluble gene product which can be readily quantified using multiwell plate spectrophotometers or luminometers. We have investigated RAW-Blue ISG™ and B16-Blue IFNα/β™ cells (InvivoGen) which produce secreted embryonic alkaline phosphatase (SEAP) as a RGA to measure Interferon alpha (IFNα) and beta (IFNβ). These cells showed a log-linear response over 4 logs of IFN concentration between 10 and 100,000 Units/ml (U/ml). Concentration dependent responses could be observed as early as 6 h but greater sensitivity was obtained at 24 h. Neutralizing antibodies to IFNα and IFNβ reduced the response to baseline. As proof of principle supernatants from RAW 264.7 (murine macrophage; parental cell line) infected with 1 multiplicity of infection (moi) of influenza A virus (X31/H3N2) were used as test samples. Pre-treatment of the supernatant with anti-IFNα failed to reduce the cell response but it was reduced to background by anti-IFNβ. The high level of IFNβ but very low level of IFNα was confirmed by ELISA. Availability, ease of use and maintenance, and possible cost savings make application of this reporter gene cell approach a valuable alternative to other methods for measuring Type I interferon.
稳定转染对 I 型干扰素（IFN）有反应的基因的细胞系可能是酶联免疫吸附测定（ELISA）或基于测试细胞系对病毒感染的抗性的测定的有用替代方法，这些测定使用细胞死亡或感染终点。越来越多的商业细胞系可用于报告基因测定（RGAs），这些细胞系对 IFN 暴露有反应。这些细胞产生一种可溶的基因产物，可使用多微孔板分光光度计或发光计轻松定量。我们研究了 RAW-Blue ISG™ 和 B16-Blue IFNα/β™细胞（InvivoGen），它们产生分泌型碱性磷酸酶（SEAP）作为 RGA 来测量干扰素 alpha（IFNα）和 beta（IFNβ）。这些细胞在 10 到 100,000 单位/毫升（U/ml）之间的 IFN 浓度范围内表现出 4 个对数的对数线性反应。早在 6 小时就可以观察到浓度依赖性反应，但在 24 小时时获得了更高的灵敏度。IFNα 和 IFNβ 的中和抗体将反应降低到基线。作为原理证明，用 1 感染复数（moi）感染流感 A 病毒（X31/H3N2）的 RAW 264.7（鼠巨噬细胞；亲本细胞系）的上清液用作测试样品。用抗 IFNα 预处理上清液未能降低细胞反应，但用抗 IFNβ 将其降低到背景水平。通过 ELISA 证实了高水平的 IFNβ 和非常低水平的 IFNα。可用性、易用性和维护性以及可能的成本节约使这种报告基因细胞方法成为测量 I 型干扰素的其他方法的有价值的替代方法。
