Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

• Published in: Talanta (Oxford) 2004-12, Vol.64 (5), p.1160-1168
• Main Authors: Soh, Nobuaki, Nishiyama, Hideshi, Asano, Yasukazu, Imato, Toshihiko, Masadome, Takashi, Kurokawa, Youichi
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Chemical and thermal methods; Chemiluminescence; Chemistry; Exact sciences and technology; Immunoassay; Magnetic microbeads; Miscellaneous; Sequential injection; Vitellogenin
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2004.06.001

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 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL −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 2 ng mL −1.