{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:16:48Z","timestamp":1760239008718,"version":"build-2065373602"},"reference-count":39,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,9,19]],"date-time":"2020-09-19T00:00:00Z","timestamp":1600473600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"2019-2020 Strategic Research Promotion of Yokohama City University","award":["SK2807"],"award-info":[{"award-number":["SK2807"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB\/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.<\/jats:p>","DOI":"10.3390\/microorganisms8091437","type":"journal-article","created":{"date-parts":[[2020,9,19]],"date-time":"2020-09-19T07:06:09Z","timestamp":1600499169000},"page":"1437","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3"],"prefix":"10.3390","volume":"8","author":[{"given":"Keiko","family":"Goto","sequence":"first","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"},{"name":"Ibaraki Prefectural Institute of Public Health, Ibaraki 310-0852, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yutaro","family":"Yamaoka","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"},{"name":"Life Science Laboratory, Technology and Development Division, Kanto Chemical Co., Inc., Kanagawa 259-1146, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hajera","family":"Khatun","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kei","family":"Miyakawa","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mayuko","family":"Nishi","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noriko","family":"Nagata","sequence":"additional","affiliation":[{"name":"Ibaraki Prefectural Institute of Public Health, Ibaraki 310-0852, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Toshikazu","family":"Yanaoka","sequence":"additional","affiliation":[{"name":"Ibaraki Prefectural Institute of Public Health, Ibaraki 310-0852, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hirokazu","family":"Kimura","sequence":"additional","affiliation":[{"name":"Department of Health Science, Gunma Paz University Graduate School, Gunma 370-0006, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Akihide","family":"Ryo","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Yokohama City University School of Medicine, Kanagawa 236-0004, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Olijve, L., Jennings, L., and Walls, T. (2018). Human Parechovirus: An Increasingly Recognized Cause of Sepsis-Like Illness in Young Infants. Clin. Microbiol. Rev., 31.","DOI":"10.1128\/CMR.00047-17"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.meegid.2015.02.003","article-title":"Detection and characterization of a novel human parechovirus genotype in Thailand","volume":"31","author":"Chuchaona","year":"2015","journal-title":"Infect. Genet. Evol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Ito, M., Yamashita, T., Tsuzuki, H., Takeda, N., and Sakae, K. (2004). Isolation and identification of a novel human parechovirus. J. Gen. Virol., 85.","DOI":"10.1099\/vir.0.19456-0"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Fernandez-Garcia, M.D., Simon-Loriere, E., Kebe, O., Sakuntabhai, A., and Ndiaye, K. (2020). Identification and molecular characterization of the first complete genome sequence of Human Parechovirus type 15. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-63467-w"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1007\/s00431-016-2725-7","article-title":"Enterovirus and parechovirus infection in children: A brief overview","volume":"175","author":"Rossen","year":"2016","journal-title":"Eur. J. Pediatr."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1093\/cid\/ciu784","article-title":"Sepsis-like disease in infants due to human parechovirus type 3 during an outbreak in Australia","volume":"60","author":"Khatami","year":"2015","journal-title":"Clin. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"12075","DOI":"10.1038\/s41598-017-12458-5","article-title":"Strain-dependent neutralization reveals antigenic variation of human parechovirus 3","volume":"7","author":"Karelehto","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1097\/INF.0b013e3181fbefc8","article-title":"Human parechovirus 3 causing sepsis-like illness in children from midwestern United States","volume":"30","author":"Selvarangan","year":"2011","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"103","DOI":"10.3201\/eid1101.040606","article-title":"Human parechovirus 3 and neonatal infections","volume":"11","author":"Boivin","year":"2005","journal-title":"Emerg. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1097\/INF.0b013e318194596a","article-title":"Human parechovirus-3 infection: Emerging pathogen in neonatal sepsis","volume":"28","author":"Levorson","year":"2009","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1111\/ped.13467","article-title":"Prevalence and characteristics of human parechovirus and enterovirus infection in febrile infants","volume":"60","author":"Sano","year":"2018","journal-title":"Pediatr. Int."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Harvala, H., Calvert, J., Van Nguyen, D., Clasper, L., Gadsby, N., Molyneaux, P., Templeton, K., McWilliams Leitch, C., and Simmonds, P. (2014). Comparison of diagnostic clinical samples and environmental sampling for enterovirus and parechovirus surveillance in Scotland, 2010 to 2012. Euro. Surveill., 19.","DOI":"10.2807\/1560-7917.ES2014.19.15.20772"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e20152848","DOI":"10.1542\/peds.2015-2848","article-title":"Parechovirus Encephalitis and Neurodevelopmental Outcomes","volume":"137","author":"Britton","year":"2016","journal-title":"Pediatrics"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1086\/649863","article-title":"Infant deaths associated with human parechovirus infection in Wisconsin","volume":"50","author":"Sedmak","year":"2010","journal-title":"Clin. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1002\/ana.21445","article-title":"Human parechovirus causes encephalitis with white matter injury in neonates","volume":"64","author":"Groenendaal","year":"2008","journal-title":"Ann. Neurol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1007\/s00431-014-2483-3","article-title":"Characteristics and outcomes of human parechovirus infection in infants (2008\u20132012)","volume":"174","author":"Vergnano","year":"2015","journal-title":"Eur. J. Pediatr."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/j.jiac.2017.04.009","article-title":"Human parechovirus type 3 infection: An emerging infection in neonates and young infants","volume":"23","author":"Aizawa","year":"2017","journal-title":"J. Infect. Chemother."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Mizuta, K., Yamakawa, T., Kurokawa, K., Chikaoka, S., Shimizu, Y., Itagaki, T., Katsushima, F., Katsushima, Y., Ito, S., and Aoki, Y. (2016). Epidemic myalgia and myositis associated with human parechovirus type 3 infections occur not only in adults but also in children: Findings in Yamagata, Japan, 2014. Epidemiol. Infect., 144.","DOI":"10.1017\/S0950268815002873"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Miyazaki, M., Hara, K., Takayoshi, T., Kawase, T., Nakagawa, Y., Arai, T., Sugimoto, T., Nishiyama, K., Gonzalez, G., and Hanaoka, N. (2020). Epidemic Myalgia Associated with Human Parechovirus Type 3 Infection. Intern. Med., 59.","DOI":"10.2169\/internalmedicine.3326-19"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.yapd.2011.03.008","article-title":"The human Parechoviruses: An overview","volume":"58","author":"Romero","year":"2011","journal-title":"Adv. Pediatr."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.jcv.2013.05.019","article-title":"Epidemic myalgia associated with human parechovirus type 3 infection among adults occurs during an outbreak among children: Findings from Yamagata, Japan, in 2011","volume":"58","author":"Mizuta","year":"2013","journal-title":"J. Clin. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Nakamura, K., Saito, K., Hara, Y., Aoyagi, T., Kitakawa, K., Abe, Y., Takemura, H., Ikeda, F., Kaku, M., and Kanemitsu, K. (2018). Severe epidemic myalgia with an elevated level of serum interleukin-6 caused by human parechovirus type 3: A case report and brief review of the literature. BMC Infect. Dis., 18.","DOI":"10.1186\/s12879-018-3284-5"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1007\/s10096-013-1906-9","article-title":"Detection of human enteroviruses and parechoviruses as part of the national enterovirus surveillance in the Netherlands, 1996-2011","volume":"32","author":"Koopmans","year":"2013","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Domanska, A., Flatt, J.W., Jukonen, J.J.J., Geraets, J.A., and Butcher, S.J. (2019). A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody. J. Virol., 93.","DOI":"10.1128\/JVI.01597-18"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"11387","DOI":"10.1038\/ncomms11387","article-title":"Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis","volume":"7","author":"Shakeel","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1038\/s41467-016-0011-z","article-title":"Genomic RNA folding mediates assembly of human parechovirus","volume":"8","author":"Shakeel","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Chen, B.C., Chang, J.T., Huang, T.S., Chen, J.J., Chen, Y.S., Jan, M.W., and Chang, T.H. (2018). Parechovirus A Detection by a Comprehensive Approach in a Clinical Laboratory. Viruses, 10.","DOI":"10.3390\/v10120711"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Abed, Y., Wolf, D., Dagan, R., and Boivin, G. (2007). Development of a serological assay based on a synthetic peptide selected from the VP0 capsid protein for detection of human parechoviruses. J. Clin. Microbiol., 45.","DOI":"10.1128\/JCM.02432-06"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"509","DOI":"10.3389\/fmicb.2016.00509","article-title":"Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen","volume":"7","author":"Yamaoka","year":"2016","journal-title":"Front. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1111\/1348-0421.12662","article-title":"Production and characterization of monoclonal antibodies specific for major capsid VP1 protein of trichodysplasia spinulosa-associated polyomavirus","volume":"62","author":"Khatun","year":"2018","journal-title":"Microbiol. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"O640","DOI":"10.1111\/1469-0691.12542","article-title":"Clinical relevance of positive human parechovirus type 1 and 3 PCR in stool samples","volume":"20","author":"Wildenbeest","year":"2014","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e51","DOI":"10.1016\/S1473-3099(18)30288-3","article-title":"Strategies to improve detection and management of human parechovirus infection in young infants","volume":"19","author":"Kadambari","year":"2019","journal-title":"Lancet Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"208","DOI":"10.3389\/fmicb.2014.00208","article-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","volume":"5","author":"Matsunaga","year":"2014","journal-title":"Front. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1136\/mp.53.3.111","article-title":"Monoclonal antibodies","volume":"53","author":"Nelson","year":"2000","journal-title":"Mol. Pathol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1186\/s12985-019-1222-9","article-title":"Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection","volume":"16","author":"Sirivichayakul","year":"2019","journal-title":"Virol. J."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"12575","DOI":"10.1128\/JVI.79.19.12575-12583.2005","article-title":"Identification of two N-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble CD4","volume":"79","author":"Pikora","year":"2005","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1515\/cclm-2017-0583","article-title":"Measurement of sirolimus concentrations in human blood using an automated electrochemiluminescence immunoassay (ECLIA): A multicenter evaluation","volume":"56","author":"Stove","year":"2018","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.jviromet.2011.08.017","article-title":"Development of a highly sensitive immunochromatographic detection kit for H5 influenza virus hemagglutinin using silver amplification","volume":"178","author":"Wada","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1002\/elps.201900059","article-title":"Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP)","volume":"40","author":"Mishra","year":"2019","journal-title":"Electrophoresis"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/8\/9\/1437\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:11:37Z","timestamp":1760177497000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/8\/9\/1437"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,19]]},"references-count":39,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,9]]}},"alternative-id":["microorganisms8091437"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms8091437","relation":{},"ISSN":["2076-2607"],"issn-type":[{"type":"electronic","value":"2076-2607"}],"subject":[],"published":{"date-parts":[[2020,9,19]]}}}