High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence
| Autor: | Koji Enomoto, Akiko Araki, Nakajima Toshihiro, Goro Kominami, Ryuji Suzuki, Marc Preaudat, Hiroshi Takemoto, Patrick Seguin, Keiji Dohi, Gérard Mathis, Hideki Ohta |
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| Rok vydání: | 2002 |
| Předmět: |
Streptavidin
Detection limit Interleukin-13 Miniaturization Chromatography Allophycocyanin medicine.diagnostic_test Chemistry High-throughput screening Fluoroimmunoassay Clinical Biochemistry Antibodies Monoclonal Pharmaceutical Science Fluorescence Cell Line Analytical Chemistry Killer Cells Natural Microtiter plate chemistry.chemical_compound Biotinylation Immunoassay Drug Discovery medicine Humans Spectroscopy |
| Zdroj: | Journal of Pharmaceutical and Biomedical Analysis. 28:73-79 |
| ISSN: | 0731-7085 |
| Popis: | A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF®) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening. |
| Databáze: | OpenAIRE |
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