title: Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen.
abstract: Protein nativity is one of the most critical factors for the quality of antigens used as immunogens and the reactivities of the resultant antibodies. The preparation and purification of native viral antigens in conventional cell-based protein expression systems are often accompanied by technical hardships. These challenges are attributable mainly to protein aggregation and insolubility during expression and purification, as well as to very low expression levels associated with the toxicity of some viral proteins. Here, we describe a novel approach for the production of monoclonal antibodies (mAbs) against nucleocapsid protein (NP) of the Middle East respiratory syndrome coronavirus (MERS-CoV). Using a wheat germ cell-free protein synthesis system, we successfully prepared large amounts of MERS-CoV NP antigen in a state that was highly soluble and intact for immunization. Following mouse immunization and hybridoma generation, we selected seven hybridoma clones that produced mAbs with exclusive reactivity against MERS-CoV NP. Epitope mapping and subsequent bioinformatic analysis revealed that these mAbs recognized epitopes located within relatively highly conserved regions of the MERS-CoV amino-acid sequence. Consistently, the mAbs exhibited no obvious cross-reactivity with NPs derived from other related viruses, including SARS coronavirus. After determining the optimal combinations of these mAbs, we developed an enzyme-linked immunosorbent assay and a rapid immunochromatographic antigen detection test that can be reliably used for laboratory diagnosis of MERS-CoV. Thus, this study provides strong evidence that the wheat germ cell-free system is useful for the production of diagnostic mAbs against emerging pathogens.
authors.affiliation: Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc.Isehara, Japan.
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Yamaoka
authors.affiliation: Department of Virology III, National Institute of Infectious Diseases Musashimurayama, Japan.
authors.forename: Shutoku
authors.initials: S
authors.lastname: Matsuyama
authors.affiliation: Department of Virology I, National Institute of Infectious Diseases Musashimurayama, Japan.
authors.forename: Shuetsu
authors.initials: S
authors.lastname: Fukushi
authors.affiliation: Department of Microbiology, School of Medicine, Yokohama City University Yokohama, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsunaga
authors.affiliation: Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Division of Virology, Kawasaki City Institute for Public HealthKawasaki, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsushima
authors.affiliation: Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kuroyama
authors.affiliation: Infectious Disease Surveillance Center, National Institute of Infectious Diseases Musashimurayama, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kimura
authors.affiliation: Department of Virology III, National Institute of Infectious Diseases Musashimurayama, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Takeda
authors.affiliation: Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Chimuro
authors.affiliation: Department of Microbiology, School of Medicine, Yokohama City University Yokohama, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Ryo
keywords: MERS-coronavirus; antigen; cell-free protein synthesis; detection; diagnosis; monoclonal antibody; nucleocapsid