A Nitric Oxide Production Bioassay for Interferon-gamma

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.