TY - JOUR
T1 - IFN-γ bioassay
T2 - Development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice
AU - Malu, Shruti
AU - Srinivasan, Shrividhya
AU - Maiti, Prasanta Kumar
AU - Rajagopal, Deepa
AU - John, Beena
AU - Nandi, Dipankar
N1 - Funding Information:
We thank Prof. R. Manjunath and Dr. V. Bal for their comments on the manuscript and encouragement. We appreciate the assistance of Veerupaxagouda, Y. Kavitha, P. Francis, S. Mukherjee and D. Chandu with some experiments and help in the preparation of this manuscript. This work was possible due to a grant from the Council of Scientific and Industrial Research, Govt. of India. PKM was supported by postdoctoral fellowships from DBT and CSIR.
PY - 2003/1/15
Y1 - 2003/1/15
N2 - Interferon-γ (IFN-γ) 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-γ 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-γ in the linear range of ∼0.03-0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-α/β and tumor necrosis factor-α (TNF-α), either alone or in combination with IFN-γ, 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-γ 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-γ in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-γ amounts in supernatants of activated T cells. The increase in IFN-γ production by activated T cells in response to CD28 costimulation was ∼3-fold by this bioassay and ∼5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-γ.
AB - Interferon-γ (IFN-γ) 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-γ 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-γ in the linear range of ∼0.03-0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-α/β and tumor necrosis factor-α (TNF-α), either alone or in combination with IFN-γ, 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-γ 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-γ in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-γ amounts in supernatants of activated T cells. The increase in IFN-γ production by activated T cells in response to CD28 costimulation was ∼3-fold by this bioassay and ∼5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-γ.
KW - Bioassay
KW - Interferon-γ
KW - Nitric oxide
KW - Peritoneal exudate cells
UR - https://www.scopus.com/pages/publications/0037437911
UR - https://www.scopus.com/pages/publications/0037437911#tab=citedBy
U2 - 10.1016/S0022-1759(02)00424-6
DO - 10.1016/S0022-1759(02)00424-6
M3 - Article
C2 - 12505712
AN - SCOPUS:0037437911
SN - 0022-1759
VL - 272
SP - 55
EP - 65
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -