title: Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.
abstract: A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Hirakawa
authors.forename: Masaaki
authors.initials: M
authors.lastname: Katayama
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Soh
authors.forename: Koji
authors.initials: K
authors.lastname: Nakano
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Imato
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis; D000941:Antigens / Q000032:analysis*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D055767:Immobilized Proteins; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D008280:Magnetics; D008863:Microspheres; D012680:Sensitivity and Specificity; D014819:Vitellogenins / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D055767:Immobilized Proteins; D014819:Vitellogenins