High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.