title: Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody.
abstract: Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.
authors.affiliation: Department of Microbiology, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsunaga
authors.affiliation: Department of Microbiology, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Kawakami
authors.affiliation: Department of Microbiology, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Matsuo
authors.affiliation: Proteome Analysis Center, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Okayama
authors.affiliation: Gunma Prefectural Institute of Public Health and Environmental Sciences Gunma, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tsukagoshi
authors.affiliation: Department of Microbiology, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kudoh
authors.affiliation: Kawasaki City Health and Safety Research Center Kanagawa, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsushima
authors.affiliation: Kawasaki City Health and Safety Research Center Kanagawa, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Shimizu
authors.affiliation: Kawasaki City Health and Safety Research Center Kanagawa, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okabe
authors.affiliation: Proteome Analysis Center, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore Singapore.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Infectious Disease Surveillance Center, National Institute of Infectious Diseases Tokyo, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kimura
authors.affiliation: Department of Microbiology, Yokohama City University School of Medicine Kanagawa, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Ryo
keywords: cell-free protein synthesis; human parainfluenza virus 3; monoclonal antibody; proteomics