Matsukuma Eiji, Kato Zenichiro, Omoya Kentaro, Hashimoto Kazuyuki, Li Ailian, Yamamoto Yutaka, Ohnishi Hidenori, Hiranuma Hidenori, Komine Hisakazu, Kondo Naomi
Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
Allergol Int. 2006 Mar;55(1):49-54. doi: 10.2332/allergolint.55.49.
Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples.
We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA.
We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA.
The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.
人干扰素-γ（hIFN-γ）由淋巴细胞产生，具有多种生物学特性。hIFN-γ的检测广泛应用于过敏性或自身免疫性疾病的各种免疫反应。酶联免疫吸附测定（ELISA）是一种用于定量细胞代谢产物或细胞因子的成熟免疫测定方法。ELISA需要许多孵育和洗涤步骤，实际上并不适合大量样品的筛选。
我们开发了一种用于检测hIFN-γ的荧光连接免疫吸附测定（FLISA）方法。我们测量了白细胞介素（IL）-18结合蛋白和抗IL-18单克隆抗体对hIFN-γ产生的50%抑制浓度（IC50）值。将FLISA描述的IC50与ELISA的进行比较。
我们开发了一种使用别藻蓝蛋白（APC）荧光蛋白测量hIFN-γ的新系统，并将其与之前使用Cy5.5的方法进行比较。所提出的FLISA的变异系数比ELISA小，ELISA和FLISA测量相同样品的变异系数平均值分别为11.1%和3.8%，这表明FLISA中常产生非特异性结果的边缘效应可能比ELISA小。
所提出的改进FLISA系统非常适合高效测量hIFN-γ。这种均相和多重方法将成为药物发现研究高通量筛选的有力工具。
