Electrochemical sandwich immunoassay for vitellogenin by sequential injection analysis using antibody immobilized magnetic microbeads

Koji Hirakawa, Masaaki Katayama, Nobuaki Soh, Koji Nakano, Hiroki Ohura, Sumio Yamasaki, Toshihiko Imato

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

A rapid and sensitive sandwich immunoassay based on a sequential injection analysis (SIA) for the determination of carp vitellogenin (Vg) is described. The SIA system was constructed from a syringe pump, a multiposition valve, a flow-through type immunoreaction cell equipped with a magnet and an amperometric detector. Magnetic microbeads immobilized with an anti-Vg monoclonal antibody (primary antibody) were used as a solid support. The primary antibody was immobilized on magnetic microbeads by coupling the primary antibody with a polylactic acid-layer, which was coated on the surface of the magnetic beads, after activated with N-hydroxysuccinimide. The introduction, trapping and flushing out of the magnetic microbeads in the immunoreaction cell were controlled by the magnet and the flow of the carrier solution. After the primary antibody-immobilized magnetic beads were introduced and trapped in the immunoreaction cell, a Vg sample solution, an alkaline phosphatase (AP)-labeled anti-Vg polyclonal antibody (secondary antibody) solution and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell based on an SIA programmed sequence. Vg was determined by the electrochemical detection of p-aminophenol (PAP), an enzymatic product of PAPP via the action by AP labeled on the secondary antibody. A solution containing PAP, which was generated in the immunoreaction cell and transiently held in a holding coil, was transported to the amperometric detector and the oxidation current of PAP on a working electrode applied at +0.20 V was measured. A sigmoidal calibration curve was obtained in the concentration range from 1 ppb to 500 ppb in a plot of oxidation current against the logarithm of the Vg concentration. The lower detection limit of the immunoassay was about 2-3 ppb. The time required for an analysis was ca. 15 min/sample.

Original languageEnglish
Pages (from-to)1297-1305
Number of pages9
JournalElectroanalysis
Volume18
Issue number13-14
DOIs
Publication statusPublished - Jul 1 2006

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Immobilized Antibodies
Vitellogenins
Antibodies
Phosphatases
Magnets
Alkaline Phosphatase
Phosphates
Detectors
Syringes
Oxidation
Monoclonal antibodies
Monoclonal Antibodies
Pumps
Calibration
Electrodes
Acids
4-aminophenol

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Electrochemistry

Cite this

Electrochemical sandwich immunoassay for vitellogenin by sequential injection analysis using antibody immobilized magnetic microbeads. / Hirakawa, Koji; Katayama, Masaaki; Soh, Nobuaki; Nakano, Koji; Ohura, Hiroki; Yamasaki, Sumio; Imato, Toshihiko.

In: Electroanalysis, Vol. 18, No. 13-14, 01.07.2006, p. 1297-1305.

Research output: Contribution to journalArticle

Hirakawa, Koji ; Katayama, Masaaki ; Soh, Nobuaki ; Nakano, Koji ; Ohura, Hiroki ; Yamasaki, Sumio ; Imato, Toshihiko. / Electrochemical sandwich immunoassay for vitellogenin by sequential injection analysis using antibody immobilized magnetic microbeads. In: Electroanalysis. 2006 ; Vol. 18, No. 13-14. pp. 1297-1305.
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abstract = "A rapid and sensitive sandwich immunoassay based on a sequential injection analysis (SIA) for the determination of carp vitellogenin (Vg) is described. The SIA system was constructed from a syringe pump, a multiposition valve, a flow-through type immunoreaction cell equipped with a magnet and an amperometric detector. Magnetic microbeads immobilized with an anti-Vg monoclonal antibody (primary antibody) were used as a solid support. The primary antibody was immobilized on magnetic microbeads by coupling the primary antibody with a polylactic acid-layer, which was coated on the surface of the magnetic beads, after activated with N-hydroxysuccinimide. The introduction, trapping and flushing out of the magnetic microbeads in the immunoreaction cell were controlled by the magnet and the flow of the carrier solution. After the primary antibody-immobilized magnetic beads were introduced and trapped in the immunoreaction cell, a Vg sample solution, an alkaline phosphatase (AP)-labeled anti-Vg polyclonal antibody (secondary antibody) solution and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell based on an SIA programmed sequence. Vg was determined by the electrochemical detection of p-aminophenol (PAP), an enzymatic product of PAPP via the action by AP labeled on the secondary antibody. A solution containing PAP, which was generated in the immunoreaction cell and transiently held in a holding coil, was transported to the amperometric detector and the oxidation current of PAP on a working electrode applied at +0.20 V was measured. A sigmoidal calibration curve was obtained in the concentration range from 1 ppb to 500 ppb in a plot of oxidation current against the logarithm of the Vg concentration. The lower detection limit of the immunoassay was about 2-3 ppb. The time required for an analysis was ca. 15 min/sample.",
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