IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice

Malu, Shruti and Srinivasan, Shrividhya and Maiti, Prasanta Kumar and Rajagopal, Deepa and John, Beena and Nandi, Dipankar (2003) IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice. In: Journal of Immunological Methods, 272 (1-2). pp. 55-65.

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Abstract

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 similar to 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 NO3- and H2O2 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 similar to3-fold by this bioassay and similar to5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma.

Item Type:	Journal Article
Publication:	Journal of Immunological Methods
Publisher:	Elsevier Science
Additional Information:	Copyright of this article belongs to Elsevier Science.
Keywords:	Interferon-g;Nitric oxide;Peritoneal exudate cells;Bioassay.
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	17 Aug 2009 06:38
Last Modified:	20 Jul 2011 05:47
URI:	http://eprints.iisc.ac.in/id/eprint/17056

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