title: Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads.
abstract: A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2-100ngmL(-1)) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2ngmL(-1).
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Soh
authors.forename: Hideshi
authors.initials: H
authors.lastname: Nishiyama
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Asano
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Imato
authors.forename: Takashi
authors.initials: T
authors.lastname: Masadome
authors.forename: Youichi
authors.initials: Y
authors.lastname: Kurokawa