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.
Kim
Y M
YM
Department of Surgery and Pharmacology, University of Pittsburgh, School of Medicine, PA 15213, USA.
Son
K
K
D000818:Animals; D002277:Carcinoma / Q000737:chemistry; D002452:Cell Count; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007371:Interferon-gamma / Q000032:analysis* / Q000097:blood; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D010051:Ovarian Neoplasms / Q000737:chemistry; D010084:Oxidation-Reduction; D011112:Polymyxin B / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D008070:Lipopolysaccharides; D009569:Nitric Oxide; D007371:Interferon-gamma; D011112:Polymyxin B