A new, simple, bioassay for human IFN-gamma.
IFN-gamma induces the production of N-formyl-kynurenine from L-tryptophan in various cell types by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO). The IFN-gamma induced IDO activity in the glioblastoma cell line 86HG39 and cells of clone 2D9 derived from this cell line was found to be greater than that in Hela cells and U373MG cells. Consequently 2D9 cells were used in all subsequent experiments. The determination of kynurenine in the supernatant of IFN-gamma activated cells was performed photometrically using a microplate reader. It was found that the amount of kynurenine produced was directly proportional to the amount of IFN-gamma used to activate cells. The detection limit for IFN-gamma of this assay was 20 U/ml. The induction of L-tryptophan degradation was specific for IFN-gamma since neither IFN-alpha, IFN-beta, IL-1, IL-2, IL-6, GM-CSF nor TNF alpha induced the production of detectable amounts of kynurenine by 86HG39 and 2D9 cells. Furthermore, a mab directed against IFN-gamma was able to completely block the IFN-gamma induced IDO activation. This bioassay was used to determine the IFN-gamma content of supernatants harvested from toxoplasma antigen specific human T cell lines and clones. This assay gave reproducible results which correlated well with the IFN-gamma content detected in the same samples using a commercially available ELISA kit. Furthermore in the case of T cell supernatant stimulated 2D9 cells a mab directed against IFN-gamma was able to completely block IDO induction. We conclude that the measurement of kynurenine production induced by IFN-gamma can be used to determinate IFN-gamma content. This is a simple bioassay which can be performed with standard laboratory equipment.
Däubener
W
W
Institut für Med. Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Wanagat
N
N
Pilz
K
K
Seghrouchni
S
S
Fischer
H G
HG
Hadding
U
U
D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007371:Interferon-gamma / Q000032:analysis*; D007737:Kynurenine / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000378:metabolism
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007737:Kynurenine; D007371:Interferon-gamma; D014366:Tryptophan Oxygenase