title: Rapid direct determination using combined separation by prepared immunomagnetic and flow cytometry of Flavobacterium psychrophilum.
abstract: Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD), was originally isolated from coho salmon Oncorhychus kisutch in the USA. Bacterial cold-water disease has since been spreading throughout Japan and has caused serious damage to populations of ayu Plecoglossus altivel in many farms and rivers. The rapid method of detecting for F. psuchrophilum is requested, however, traditional methods are laborious because of complicated assay procedures. In this study, a rapid method of detecting F. psychrophilum was developed using a modified method of flow cytometry (FCM) analysis and immunomagnetic separation (IMS). Magnetic iron, in small particles, was prepared by the reaction of a mixture of ferric and ferrous ions under alkaline conditions. The particles were coated with antiserum against F. psychrophilum by dextran. Polyclonal antibodies (anti-F. psychrophilum) conjugated with fluorescein isothiocyanate (FITC) were reacted with F. psychrophilum, and then prepared immunomagnetic were applied using IMS, followed by FCM determination. A good correlation was observed between the cell numbers determined by the FCM method and the traditional method in the range of 10(2)-10(8) cells ml(-1). The FCM analysis could count cells within 1min, and the total analysis time, including sample preparation, was less than 2 h.
authors.affiliation: Faculty of Marine Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Hibi
authors.forename: Kohji
authors.initials: K
authors.lastname: Mitsubayashi
authors.forename: Hideo
authors.initials: H
authors.lastname: Fukuda
authors.forename: Hideki
authors.initials: H
authors.lastname: Ushio
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Hayashi
authors.forename: Huifeng
authors.initials: H
authors.lastname: Ren
authors.forename: Hideaki
authors.initials: H
authors.lastname: Endo
mesh_terms: D002469:Cell Separation; D005417:Flavobacterium / Q000302:isolation & purification*; D005434:Flow Cytometry; D018189:Immunomagnetic Separation; D007501:Iron; D053768:Metal Nanoparticles
chemical_list: D007501:Iron