Document Detail


A nitric oxide production bioassay for interferon-gamma.
MedLine Citation:
PMID:  8946016     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Y M Kim; K Son
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of immunological methods     Volume:  198     ISSN:  0022-1759     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  1996 Nov 
Date Detail:
Created Date:  1997-01-07     Completed Date:  1997-01-07     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  203-9     Citation Subset:  IM    
Affiliation:
Department of Surgery and Pharmacology, University of Pittsburgh, School of Medicine, PA 15213, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Carcinoma / chemistry
Cell Count
Cell Line
Dose-Response Relationship, Immunologic
Female
Humans
Immunoassay / methods*
Interferon-gamma / analysis*,  blood
Lipopolysaccharides / pharmacology
Macrophages / cytology
Mice
Nitric Oxide / analysis,  antagonists & inhibitors,  biosynthesis*
Ovarian Neoplasms / chemistry
Oxidation-Reduction
Polymyxin B / pharmacology
Rats
Tumor Cells, Cultured
Chemical
Reg. No./Substance:
0/Lipopolysaccharides; 10102-43-9/Nitric Oxide; 1404-26-8/Polymyxin B; 82115-62-6/Interferon-gamma

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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