K. Enomoto et al., High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence, J BIOMOL SC, 5(4), 2000, pp. 263-268
An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-reso
lved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can b
e detected by simply adding a mixture of three reagents-biotinylated polycl
onal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclon
al antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665)
conjugated with streptavidin-and then measuring the time-resolved fluoresc
ence. 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 NK
3.3 cells and employed it in high throughput screening for IFN-gamma produc
tion inhibitors. With this screening format, IFN-gamma can be measured by d
irectly adding the above reagents to microplate wells where NK3.3 cells are
being cultured and stimulated with interleukin-12, This "in situ" immunoas
say requires only pipetting reagents, with no need to transfer the culture
supernatant to another microplate or wash the plate. Therefore, this screen
ing format makes possible full automation of cell-based immunoassay, thus r
educing cost and experimental time while increasing accuracy and throughput
.