A NEW, SIMPLE, BIOASSAY FOR HUMAN IFN-GAMMA

IFN-gamma induces the production of N-formyl-kynurenine from L-tryptop han in various cell types by the induction of the enzyme indoleamine 2 ,3-dioxygenase (IDO). The IFN-gamma induced IDO activity in the gliobl astoma 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 d etermination of kynurenine in the supernatant of IFN-gamma activated c ells was performed photometrically using a microplate reader. It was f ound 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-tryptopha n 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 IF N-gamma induced IDO activation. This bioassay was used to determine th e IFN-gamma content of supernatants harvested from toxoplasma antigen specific human T cell lines and clones. This assay gave reproducible r esults which correlated well with the IFN-gamma content detected in th e same samples using a commercially available ELISA kit. Furthermore i n the case of T cell supernatant stimulated 2D9 cells a mab directed a gainst IFN-gamma was able to completely block IDO induction. We conclu de that the measurement of kynurenine production induced by IFN-gamma can be used to determinate IFN-gamma content. This is a simple bioassa y which can be performed with standard laboratory equipment.