Electrochemical Sandwich Immunoassay for Vitellogenin by Sequential Injection Analysis Using Antibody Immobilized Magnetic Microbeads

Abstract

AbstractA 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.