Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

Nobuaki Soh, Hideshi Nishiyama, Yasukazu Asano, Toshihiko Imato, Takashi Masadome, Youichi Kurokawa

Research output: Contribution to journalArticle

44 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)1160-1168
Number of pages9
JournalTalanta
Volume64
Issue number5 SPEC. ISS.
DOIs
Publication statusPublished - 2004 Dec 15
Externally publishedYes

Fingerprint

Vitellogenins
Chemiluminescence
Antibodies
Horseradish Peroxidase
Magnets
Luminol
Monoclonal Antibodies
Immobilized Antibodies
Photomultipliers
Sepharose
Hydrogen Peroxide
Calibration
Detectors

Keywords

  • Chemiluminescence
  • Immunoassay
  • Magnetic microbeads
  • Sequential injection
  • Vitellogenin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy

Cite this

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads. / Soh, Nobuaki; Nishiyama, Hideshi; Asano, Yasukazu; Imato, Toshihiko; Masadome, Takashi; Kurokawa, Youichi.

In: Talanta, Vol. 64, No. 5 SPEC. ISS., 15.12.2004, p. 1160-1168.

Research output: Contribution to journalArticle

Soh, N, Nishiyama, H, Asano, Y, Imato, T, Masadome, T & Kurokawa, Y 2004, 'Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads', Talanta, vol. 64, no. 5 SPEC. ISS., pp. 1160-1168. https://doi.org/10.1016/j.talanta.2004.06.001
Soh, Nobuaki ; Nishiyama, Hideshi ; Asano, Yasukazu ; Imato, Toshihiko ; Masadome, Takashi ; Kurokawa, Youichi. / Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads. In: Talanta. 2004 ; Vol. 64, No. 5 SPEC. ISS. pp. 1160-1168.
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AB - 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.

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