CYTOKINE IMMUNOTRAPPING - AN ASSAY TO STUDY THE KINETICS OF PRODUCTION AND CONSUMPTION OR DEGRADATION OF HUMAN INTERFERON-GAMMA

With the aim of determining the rate of cytokine production, we have investigated immunoassay conditions which prevent consumption/degradation. These assays, termed cytokine immunotrapping assays (CITA), are based on early capturing of cytokines secreted during cell culture by immobilised or soluble mAbs and a recently described chemiluminescent immunoassay. Here we describe assay conditions using IFN-gamma as a prototype cytokine. For production of IFN-gamma, PBMC, purified CD4(+) or CD8(+) T cells, or diluted whole blood were cultured with different T cell stimulating agents. Polystyrene macrobeads precoated with an anti-IFN-gamma mAb were put in culture and after a defined incubation period, a dimethyl acridinium ester (DMAE)-labelled second anti-IFN-gamma mAb and sodium azide were added into the culture for additional 24 h. The beads were washed and chemiluminescence signals determined in a luminometer. Trapping experiments were also performed with the beads or the soluble mAbs alone. Irrespective of the configuration, IFN-gamma concentrations measured in trapping conditions were always higher (3-20-fold) than in conventional cultures. By using the best trapping combination, i.e. both bead-mAb1 and DMAE-mAb2 added at the start of culture (single step), it was possible to detect IFN-gamma production as early as 2 h. Also, IFN-gamma secreted by less than 500 PBMC or whole blood cells could be detected within 24 h. When purified CD4(+) or CD8(+) cells were used instead of PBMC, a reduction of the trapping effect was observed. Conversely, addition of monocytes to purified T cells increased the trapping factor suggesting that a substantial amount of IFN-gamma was consumed or degraded both by CD14(+) cells as well as T cells in culture. Preliminary results show that this assay is also suitable for the early detection of IL-1 and IL-4 which are known to be more tightly regulated. Thus, the new principle described here is expected to be useful in clinical settings where both the time and amounts of material are limited to investigate the role of cytokines in particular disease.