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 language | English |
|---|---|
| Pages (from-to) | 1297-1305 |
| Number of pages | 9 |
| Journal | Electroanalysis |
| Volume | 18 |
| Issue number | 13-14 |
| DOIs | |
| Publication status | Published - Jul 1 2006 |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Electrochemical sandwich immunoassay for vitellogenin by sequential injection analysis using antibody immobilized magnetic microbeads
AU - Hirakawa, Koji
AU - Katayama, Masaaki
AU - Soh, Nobuaki
AU - Nakano, Koji
AU - Ohura, Hiroki
AU - Yamasaki, Sumio
AU - Imato, Toshihiko
PY - 2006/7/1
Y1 - 2006/7/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33746638479&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33746638479&partnerID=8YFLogxK
U2 - 10.1002/elan.200603529
DO - 10.1002/elan.200603529
M3 - Article
VL - 18
SP - 1297
EP - 1305
JO - Electroanalysis
JF - Electroanalysis
SN - 1040-0397
IS - 13-14
ER -