High Throughput Screening for Human Interferon-y Production Inhibitor Using Homogenous Time-Resolved Fluorescence

An immunoassay for interferon-γ (IFN-γ) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-γ 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-γ by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-γ secreted from NK3.3 cells and employed it in high throughput screening for IFN-γ production inhibitors. With this screening format, IFN-γ 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 scre...