title: High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence.
abstract: 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.
authors.affiliation: Developmental Research Laboratories, Shionogi & Co. Ltd., 3-1-1, Futaba-cho, Toyonaka, Osaka, Japan. koji.enomoto@shionogi.co.jp
authors.forename: Koji
authors.initials: K
authors.lastname: Enomoto
authors.forename: Akiko
authors.initials: A
authors.lastname: Araki
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nakajima
authors.forename: Hideki
authors.initials: H
authors.lastname: Ohta
authors.forename: Keiji
authors.initials: K
authors.lastname: Dohi
authors.forename: Marc
authors.initials: M
authors.lastname: Préaudat
authors.forename: Patrick
authors.initials: P
authors.lastname: Seguin
authors.forename: Gérard
authors.initials: G
authors.lastname: Mathis
authors.forename: Ryuji
authors.initials: R
authors.lastname: Suzuki
authors.forename: Goro
authors.initials: G
authors.lastname: Kominami
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takemoto
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis; D002460:Cell Line; D015200:Fluoroimmunoassay / Q000379:methods; D006801:Humans; D018793:Interleukin-13 / Q000032:analysis* / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000166:cytology / Q000378:metabolism*; D008904:Miniaturization / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D018793:Interleukin-13