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

Citation
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
Citations number
24
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Chemistry & Analysis
Journal title
JOURNAL OF BIOMOLECULAR SCREENING
ISSN journal
1087-0571 → ACNP
Volume
5
Issue
4
Year of publication
2000
Pages
263 - 268
Database
ISI
SICI code
1087-0571(200008)5:4<263:HTSFHI>2.0.ZU;2-A
Abstract
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 .