IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice.
Interferon-gamma (IFN-gamma) is an important immunomodulatory and pleiotropic cytokine produced, primarily, by activated T lymphocytes and natural killer (NK) cells. We have devised a nitric oxide (NO)-based bioassay for mouse IFN-gamma using resident peritoneal exudate cells (PECs) from C57BL/6 mice. Comparison with three existing bioassays demonstrated that this assay was very sensitive and detected IFN-gamma in the linear range of approximately 0.03-0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-alpha/beta and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination with IFN-gamma, did not greatly modulate NO levels produced by resident peritoneal exudate cells. The presence of exogenous NO(3)(-) and H(2)O(2) did not interfere with the IFN-gamma induced NO production and detection. We also showed that the effect of lipopolysaccharide (LPS), which may be present in samples, could be suppressed by the use of Polymyxin B in the bioassay. The high sensitivity of the bioassay permitted the detection of low amounts of IFN-gamma in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-gamma amounts in supernatants of activated T cells. The increase in IFN-gamma production by activated T cells in response to CD28 costimulation was approximately 3-fold by this bioassay and approximately 5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma.
Malu
Shruti
S
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Srinivasan
Shrividhya
S
Kumar Maiti
Prasanta
P
Rajagopal
Deepa
D
John
Beena
B
Nandi
Dipankar
D
D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D001681:Biological Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000032:analysis*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000096:biosynthesis*; D011112:Polymyxin B / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology
D016207:Cytokines; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D007371:Interferon-gamma; D011112:Polymyxin B