Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen.
Identifieur interne : 001045 ( Main/Exploration ); précédent : 001044; suivant : 001046Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen.
Auteurs : Yutaro Yamaoka [Japon] ; Shutoku Matsuyama [Japon] ; Shuetsu Fukushi [Japon] ; Satoko Matsunaga [Japon] ; Yuki Matsushima [Japon] ; Hiroyuki Kuroyama [Japon] ; Hirokazu Kimura [Japon] ; Makoto Takeda [Japon] ; Tomoyuki Chimuro [Japon] ; Akihide Ryo [Japon]Source :
- Frontiers in microbiology [ 1664-302X ] ; 2016.
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.
DOI: 10.3389/fmicb.2016.00509
PubMed: 27148198
Affiliations:
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Isehara, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara</wicri:regionArea><wicri:noRegion>Inc. Isehara</wicri:noRegion></affiliation></author><author><name sortKey="Ryo, Akihide" sort="Ryo, Akihide" uniqKey="Ryo A" first="Akihide" last="Ryo">Akihide Ryo</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, School of Medicine, Yokohama City University Yokohama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Microbiology, School of Medicine, Yokohama City University Yokohama</wicri:regionArea><wicri:noRegion>Yokohama City University Yokohama</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2016">2016</date><idno type="RBID">pubmed:27148198</idno><idno type="pmid">27148198</idno><idno type="doi">10.3389/fmicb.2016.00509</idno><idno type="wicri:Area/PubMed/Corpus">000C57</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000C57</idno><idno type="wicri:Area/PubMed/Curation">000C57</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">000C57</idno><idno type="wicri:Area/PubMed/Checkpoint">000C70</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">000C70</idno><idno type="wicri:Area/Ncbi/Merge">002C57</idno><idno type="wicri:Area/Ncbi/Curation">002C57</idno><idno type="wicri:Area/Ncbi/Checkpoint">002C57</idno><idno type="wicri:doubleKey">1664-302X:2016:Yamaoka Y:development:of:monoclonal</idno><idno type="wicri:Area/Main/Merge">001047</idno><idno type="wicri:Area/Main/Curation">001045</idno><idno type="wicri:Area/Main/Exploration">001045</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen.</title><author><name sortKey="Yamaoka, Yutaro" sort="Yamaoka, Yutaro" uniqKey="Yamaoka Y" first="Yutaro" last="Yamaoka">Yutaro Yamaoka</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc.Isehara, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc.Isehara</wicri:regionArea><wicri:noRegion>Inc.Isehara</wicri:noRegion></affiliation></author><author><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><affiliation wicri:level="1"><nlm:affiliation>Department of Virology III, National Institute of Infectious Diseases Musashimurayama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Virology III, National Institute of Infectious Diseases Musashimurayama</wicri:regionArea><wicri:noRegion>National Institute of Infectious Diseases Musashimurayama</wicri:noRegion></affiliation></author><author><name sortKey="Fukushi, Shuetsu" sort="Fukushi, Shuetsu" uniqKey="Fukushi S" first="Shuetsu" last="Fukushi">Shuetsu Fukushi</name><affiliation wicri:level="1"><nlm:affiliation>Department of Virology I, National Institute of Infectious Diseases Musashimurayama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Virology I, National Institute of Infectious Diseases Musashimurayama</wicri:regionArea><wicri:noRegion>National Institute of Infectious Diseases Musashimurayama</wicri:noRegion></affiliation></author><author><name sortKey="Matsunaga, Satoko" sort="Matsunaga, Satoko" uniqKey="Matsunaga S" first="Satoko" last="Matsunaga">Satoko Matsunaga</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, School of Medicine, Yokohama City University Yokohama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Microbiology, School of Medicine, Yokohama City University Yokohama</wicri:regionArea><wicri:noRegion>Yokohama City University Yokohama</wicri:noRegion></affiliation></author><author><name sortKey="Matsushima, Yuki" sort="Matsushima, Yuki" uniqKey="Matsushima Y" first="Yuki" last="Matsushima">Yuki Matsushima</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Division of Virology, Kawasaki City Institute for Public HealthKawasaki, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Microbiology, School of Medicine, Yokohama City UniversityYokohama, Japan; Division of Virology, Kawasaki City Institute for Public HealthKawasaki</wicri:regionArea><wicri:noRegion>Kawasaki City Institute for Public HealthKawasaki</wicri:noRegion></affiliation></author><author><name sortKey="Kuroyama, Hiroyuki" sort="Kuroyama, Hiroyuki" uniqKey="Kuroyama H" first="Hiroyuki" last="Kuroyama">Hiroyuki Kuroyama</name><affiliation wicri:level="1"><nlm:affiliation>Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara</wicri:regionArea><wicri:noRegion>Inc. Isehara</wicri:noRegion></affiliation></author><author><name sortKey="Kimura, Hirokazu" sort="Kimura, Hirokazu" uniqKey="Kimura H" first="Hirokazu" last="Kimura">Hirokazu Kimura</name><affiliation wicri:level="1"><nlm:affiliation>Infectious Disease Surveillance Center, National Institute of Infectious Diseases Musashimurayama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Infectious Disease Surveillance Center, National Institute of Infectious Diseases Musashimurayama</wicri:regionArea><wicri:noRegion>National Institute of Infectious Diseases Musashimurayama</wicri:noRegion></affiliation></author><author><name sortKey="Takeda, Makoto" sort="Takeda, Makoto" uniqKey="Takeda M" first="Makoto" last="Takeda">Makoto Takeda</name><affiliation wicri:level="1"><nlm:affiliation>Department of Virology III, National Institute of Infectious Diseases Musashimurayama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Virology III, National Institute of Infectious Diseases Musashimurayama</wicri:regionArea><wicri:noRegion>National Institute of Infectious Diseases Musashimurayama</wicri:noRegion></affiliation></author><author><name sortKey="Chimuro, Tomoyuki" sort="Chimuro, Tomoyuki" uniqKey="Chimuro T" first="Tomoyuki" last="Chimuro">Tomoyuki Chimuro</name><affiliation wicri:level="1"><nlm:affiliation>Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Isehara Research Laboratory, Technology and Development Division, Kanto Chemical Co., Inc. Isehara</wicri:regionArea><wicri:noRegion>Inc. Isehara</wicri:noRegion></affiliation></author><author><name sortKey="Ryo, Akihide" sort="Ryo, Akihide" uniqKey="Ryo A" first="Akihide" last="Ryo">Akihide Ryo</name><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology, School of Medicine, Yokohama City University Yokohama, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Microbiology, School of Medicine, Yokohama City University Yokohama</wicri:regionArea><wicri:noRegion>Yokohama City University Yokohama</wicri:noRegion></affiliation></author></analytic><series><title level="j">Frontiers in microbiology</title><idno type="ISSN">1664-302X</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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. </div></front></TEI><affiliations><list><country><li>Japon</li></country></list><tree><country name="Japon"><noRegion><name sortKey="Yamaoka, Yutaro" sort="Yamaoka, Yutaro" uniqKey="Yamaoka Y" first="Yutaro" last="Yamaoka">Yutaro Yamaoka</name></noRegion><name sortKey="Chimuro, Tomoyuki" sort="Chimuro, Tomoyuki" uniqKey="Chimuro T" first="Tomoyuki" last="Chimuro">Tomoyuki Chimuro</name><name sortKey="Fukushi, Shuetsu" sort="Fukushi, Shuetsu" uniqKey="Fukushi S" first="Shuetsu" last="Fukushi">Shuetsu Fukushi</name><name sortKey="Kimura, Hirokazu" sort="Kimura, Hirokazu" uniqKey="Kimura H" first="Hirokazu" last="Kimura">Hirokazu Kimura</name><name sortKey="Kuroyama, Hiroyuki" sort="Kuroyama, Hiroyuki" uniqKey="Kuroyama H" first="Hiroyuki" last="Kuroyama">Hiroyuki Kuroyama</name><name sortKey="Matsunaga, Satoko" sort="Matsunaga, Satoko" uniqKey="Matsunaga S" first="Satoko" last="Matsunaga">Satoko Matsunaga</name><name sortKey="Matsushima, Yuki" sort="Matsushima, Yuki" uniqKey="Matsushima Y" first="Yuki" last="Matsushima">Yuki Matsushima</name><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><name sortKey="Ryo, Akihide" sort="Ryo, Akihide" uniqKey="Ryo A" first="Akihide" last="Ryo">Akihide Ryo</name><name sortKey="Takeda, Makoto" sort="Takeda, Makoto" uniqKey="Takeda M" first="Makoto" last="Takeda">Makoto Takeda</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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