High Throughput Screening for Human Interferon-γ Production Inhibitor Using Homogenous Time-Resolved Fluorescence
| Autor: | Gérard Mathis, Marc Preaudat, Koji Takahashi, Goro Kominami, Ryuji Suzuki, Hiroshi Takemoto, Koji Enomoto, Yuko Aono, Takashi Mitsugi |
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| Rok vydání: | 2000 |
| Předmět: |
0301 basic medicine
medicine.drug_class High-throughput screening Fluoroimmunoassay Drug Evaluation Preclinical Enzyme-Linked Immunosorbent Assay Monoclonal antibody 01 natural sciences Biochemistry Cell Line Analytical Chemistry Interferon-gamma 03 medical and health sciences medicine Humans Fluorescent Dyes Detection limit Allophycocyanin Chromatography medicine.diagnostic_test biology Chemistry Antibodies Monoclonal Robotics Reference Standards Fluorescence Molecular biology Recombinant Proteins 0104 chemical sciences 010404 medicinal & biomolecular chemistry 030104 developmental biology Polyclonal antibodies Immunoassay biology.protein Molecular Medicine Time-resolved spectroscopy Biotechnology |
| Zdroj: | SLAS Discovery. 5:263-268 |
| ISSN: | 2472-5552 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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