Kim Y M, Son K
Department of Surgery and Pharmacology, University of Pittsburgh, School of Medicine, PA 15213, USA.
J Immunol Methods. 1996 Nov 13;198(2):203-9. doi: 10.1016/s0022-1759(96)00162-7.
Interferon-gamma (IFN-gamma), produced by stimulated T lymphocytes and natural killer cells, has an antitumor and antiviral activity by inhibition of cell growth, immunomodulation, and/or activation of macrophages. Although several IFN-gamma assays have been used, there are no simple, inexpensive, and specific assays. We have developed a new bioassay for IFN-gamma which measures the concentration of nitric oxide (NO) generated by a macrophage cell line RAW264.7 stimulated with IFN-gamma. NO production, determined by nitrite (NO2-) accumulation in culture medium, was linear at IFN-gamma concentrations between 0 and 10 U/ml and logarithmically linear between 2 and 100 U/ml, when RAW cells (1 x 10(5) cells/200 microliters/well in 96 well plate) were incubated with murine recombinant IFN-gamma for 24 h. The new assay has a high sensitivity with the detection limit of 0.1-0.2 ng/ml IFN-gamma, which is similar to that of the enzyme-linked immunosorbent assay (ELISA) and antiviral assays. Other cytokines such as IFN-alpha, IFN-beta, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-2, and IL-6, either alone or in combination did not produce NO from RAW264.7. Exogenous NO2-, NO3- or NO2- plus NO3- did not interfere with the IFN-gamma-induced NO formation as well as NO2- measurement. The IFN-gamma bioactivity, measured by the RAW264.7 bioassay, expressed from the transfected human ovarian tumor cells was closely correlated with the levels of IFN-gamma protein concentration measured by the ELISA. Therefore, our new method can be applicable for monitoring IFN-gamma gene expression and accumulation in culture medium for IFN-gamma therapy.
干扰素-γ（IFN-γ）由受刺激的T淋巴细胞和自然杀伤细胞产生，通过抑制细胞生长、免疫调节和/或激活巨噬细胞而具有抗肿瘤和抗病毒活性。虽然已经使用了几种IFN-γ检测方法，但尚无简单、廉价且特异的检测方法。我们开发了一种新的IFN-γ生物检测方法，该方法可测量经IFN-γ刺激的巨噬细胞系RAW264.7产生的一氧化氮（NO）浓度。当RAW细胞（96孔板中每孔1×10⁵个细胞/200微升）与鼠重组IFN-γ孵育24小时时，通过培养基中亚硝酸盐（NO₂⁻）积累测定的NO产生在IFN-γ浓度为0至10 U/ml时呈线性，在2至100 U/ml时呈对数线性。新检测方法具有高灵敏度，检测限为0.1 - 0.2 ng/ml IFN-γ，这与酶联免疫吸附测定（ELISA）和抗病毒检测方法相似。其他细胞因子，如IFN-α、IFN-β、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-2和IL-6，单独或联合使用均不能使RAW264.7产生NO。外源性NO₂⁻、NO₃⁻或NO₂⁻加NO₃⁻均不干扰IFN-γ诱导的NO形成以及NO₂⁻测量。通过RAW264.7生物检测法测定的转染人卵巢肿瘤细胞表达的IFN-γ生物活性与通过ELISA测定的IFN-γ蛋白浓度水平密切相关。因此，我们的新方法可用于监测IFN-γ基因在培养基中的表达和积累，以用于IFN-γ治疗。
