{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T01:55:22Z","timestamp":1760234122720,"version":"build-2065373602"},"reference-count":44,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,4,16]],"date-time":"2021-04-16T00:00:00Z","timestamp":1618531200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004663","name":"Ministry of Science and Technology, Taiwan","doi-asserted-by":"publisher","award":["MOST108-2622-B-038-003-CC2, MOST 109-2320-B-075A-001, and MOST 109-2320-B-038-053"],"award-info":[{"award-number":["MOST108-2622-B-038-003-CC2, MOST 109-2320-B-075A-001, and MOST 109-2320-B-038-053"]}],"id":[{"id":"10.13039\/501100004663","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Ministry of Health and Welfare surcharge of tobacco products","award":["MOHW108-TDU-B-212-124014 and MOHW 109-TDU-B-212-134014"],"award-info":[{"award-number":["MOHW108-TDU-B-212-124014 and MOHW 109-TDU-B-212-134014"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged &lt;5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.<\/jats:p>","DOI":"10.3390\/ijms22084146","type":"journal-article","created":{"date-parts":[[2021,4,16]],"date-time":"2021-04-16T10:20:11Z","timestamp":1618568411000},"page":"4146","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9260-8467","authenticated-orcid":false,"given":"Pharaoh Fellow","family":"Mwale","sequence":"first","affiliation":[{"name":"Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"},{"name":"School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3412-7400","authenticated-orcid":false,"given":"Chi-Hsin","family":"Lee","sequence":"additional","affiliation":[{"name":"Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"},{"name":"School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peng-Nien","family":"Huang","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Pediatrics, Linkou Chang Gung Memorial Hospital, Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan 333423, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sung-Nien","family":"Tseng","sequence":"additional","affiliation":[{"name":"Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan 333323, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shin-Ru","family":"Shih","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan 333423, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hsin-Yuan","family":"Huang","sequence":"additional","affiliation":[{"name":"School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sy-Jye","family":"Leu","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110301, Taiwan"},{"name":"Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun-Ju","family":"Huang","sequence":"additional","affiliation":[{"name":"School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liao-Chun","family":"Chiang","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsinchu 300040, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8561-0582","authenticated-orcid":false,"given":"Yan-Chiao","family":"Mao","sequence":"additional","affiliation":[{"name":"Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung 407219, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei-Chu","family":"Wang","sequence":"additional","affiliation":[{"name":"Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2534-8436","authenticated-orcid":false,"given":"Yi-Yuan","family":"Yang","sequence":"additional","affiliation":[{"name":"Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"},{"name":"School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110301, Taiwan"},{"name":"Core Laboratory of Antibody Generation and Research, Taipei Medical University, Taipei 110301, Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"761","DOI":"10.3892\/br.2016.663","article-title":"Molecular epidemiology of human coxsackievirus A16 strains","volume":"4","author":"Yu","year":"2016","journal-title":"Biomed. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"360","DOI":"10.4161\/hv.27087","article-title":"Coxsackievirus A16: Epidemiology, diagnosis, and vaccine","volume":"10","author":"Mao","year":"2014","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/1743-422X-9-8","article-title":"Distribution of enteroviruses in hospitalized children with hand, foot and mouth disease and relationship between pathogens and nervous system complications","volume":"9","author":"Xu","year":"2012","journal-title":"Virol. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1097\/01.inf.0000115950.63906.78","article-title":"Fatal coxsackievirus A16 infection","volume":"23","author":"Wang","year":"2004","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_5","first-page":"R81","article-title":"Epidemiological, virological, and clinical features of an epidemic of hand, foot, and mouth disease in England and Wales","volume":"6","author":"Bendig","year":"1996","journal-title":"Commun. Dis. Rep. CDR Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.pedneo.2013.07.007","article-title":"Epidemiology of enterovirus 71 infections in Taiwan","volume":"55","author":"Chia","year":"2014","journal-title":"Pediatr. Neonatol."},{"key":"ref_7","first-page":"1537","article-title":"Circulating coxsackievirus A16 identified as recombinant type A human enterovirus, China","volume":"17","author":"Zhao","year":"2011","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1186\/1743-422X-7-94","article-title":"An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China","volume":"7","author":"Zhang","year":"2010","journal-title":"Virol. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1586\/14760584.2016.1138862","article-title":"EV71 vaccine, a new tool to control outbreaks of hand, foot and mouth disease (HFMD)","volume":"15","author":"Mao","year":"2016","journal-title":"Expert Rev. Vaccines"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"547","DOI":"10.1093\/tropej\/fmz004","article-title":"Safety comparison of two enterovirus 71 (EV71) inactivated vaccines in Yiwu, China","volume":"65","author":"Luo","year":"2019","journal-title":"J. Trop. Pediatr."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Tan, Y., and Chu, J. (2017). Sinovac EV71 vaccine: The silver bullet for hand, foot and mouth disease\u2014Or not. J. Public Health Emerg., 1.","DOI":"10.21037\/jphe.2016.12.14"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1469","DOI":"10.1056\/NEJMp1802256","article-title":"Monoclonal antibodies for emerging infectious diseases\u2014Borrowing from history","volume":"378","author":"Marston","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1097\/INF.0000000000001090","article-title":"Enterovirus Infections of the Central Nervous System in Children: An Update","volume":"35","author":"Rudolph","year":"2016","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8185","DOI":"10.1038\/srep08185","article-title":"Molecular evolution of the VP1, VP2, and VP3 genes in human rhinovirus species C","volume":"5","author":"Kuroda","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6774","DOI":"10.1073\/pnas.95.12.6774","article-title":"Antiviral agent blocks breathing of the common cold virus","volume":"95","author":"Lewis","year":"1998","journal-title":"Proc. Nat. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4143","DOI":"10.1128\/JVI.03101-14","article-title":"Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry","volume":"89","author":"Strauss","year":"2015","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1126\/science.3140379","article-title":"Single-chain antigen-binding proteins","volume":"242","author":"Bird","year":"1988","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5879","DOI":"10.1073\/pnas.85.16.5879","article-title":"Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli","volume":"85","author":"Huston","year":"1988","journal-title":"Proc. Nat. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.ejbt.2017.07.003","article-title":"Application of a single-chain fragment variable (scFv) antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas","volume":"29","author":"Xu","year":"2017","journal-title":"Electron. J. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"7724","DOI":"10.1158\/0008-5472.CAN-05-4441","article-title":"Selection of tumor-binding ligands in cancer patients with phage display libraries","volume":"66","author":"Krag","year":"2006","journal-title":"Cancer Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Unkauf, T., Miethe, S., F\u00fchner, V., Schirrmann, T., Frenzel, A., and Hust, M. (2016). Generation of recombinant antibodies against toxins and viruses by phage display for diagnostics and therapy. Protein Targeting Compounds, Springer.","DOI":"10.1007\/978-3-319-32805-8_4"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1126\/science.1205669","article-title":"A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins","volume":"333","author":"Corti","year":"2011","journal-title":"Science"},{"key":"ref_23","first-page":"3402","article-title":"Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms","volume":"52","author":"Yokota","year":"1992","journal-title":"Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2030","DOI":"10.1016\/j.vaccine.2009.01.130","article-title":"Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus","volume":"27","author":"Pyo","year":"2009","journal-title":"Vaccine"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"904","DOI":"10.1016\/j.vaccine.2009.01.110","article-title":"Passive immunization with llama single-domain antibody fragments reduces foot-and-mouth disease transmission between pigs","volume":"27","author":"Harmsen","year":"2009","journal-title":"Vaccine"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Bird, C.R., and Thorpe, R. (2009). Purification of immunoglobulin Y (IgY) from chicken eggs. The Protein Protocols Handbook, Springer.","DOI":"10.1007\/978-1-59745-198-7_189"},{"key":"ref_27","first-page":"587","article-title":"Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System","volume":"10","author":"Deng","year":"2003","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Hust, M., and Lim, T.S. (2018). Construction of Chicken Antibody Libraries. Phage Display: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-4939-7447-4"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1002\/jmv.24151","article-title":"Epidemiological and etiological characteristics of hand, foot, and mouth disease in Wuhan, China from 2012 to 2013: Outbreaks of coxsackieviruses A10","volume":"87","author":"Yang","year":"2015","journal-title":"J. Med. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Nilvebrant, J., and Sidhu, S.S. (2018). Construction of synthetic antibody phage-display libraries. Phage Display, Springer.","DOI":"10.1007\/978-1-4939-7447-4_3"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.jim.2018.06.023","article-title":"Production and application of anti-nucleoprotein IgY antibodies for influenza A virus detection in swine","volume":"461","author":"Schaefer","year":"2018","journal-title":"J. Immunol. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lee, C.-H., Leu, S.-J., Lee, Y.-C., Liu, C.-I., Lin, L.-T., Mwale, P.F., Chiang, J.-R., Tsai, B.-Y., Chen, C.-C., and Hung, C.-S. (2018). Characterization of chicken-derived single chain antibody fragments against venom of Naja naja atra. Toxins, 10.","DOI":"10.3390\/toxins10100383"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1016\/j.vaccine.2013.03.007","article-title":"Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection","volume":"31","author":"Cai","year":"2013","journal-title":"Vaccine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1186\/1743-422X-8-534","article-title":"Construction and characterization of an infectious clone of coxsackievirus A16","volume":"8","author":"Liu","year":"2011","journal-title":"Virol. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/s00705-015-2625-6","article-title":"Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2)","volume":"161","author":"Li","year":"2016","journal-title":"Arch. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2343","DOI":"10.1080\/21645515.2019.1565266","article-title":"A potential therapeutic neutralization monoclonal antibody specifically against multi-coxsackievirus A16 strains challenge","volume":"15","author":"Du","year":"2019","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_37","first-page":"1","article-title":"A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity","volume":"7","author":"Zhang","year":"2018","journal-title":"Emerg. Microbes Infect."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1002\/jmv.25065","article-title":"Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus","volume":"90","author":"Zhang","year":"2018","journal-title":"J. Med. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5285","DOI":"10.1007\/s00253-019-09761-8","article-title":"Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies","volume":"103","author":"Mwale","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.vetimm.2010.06.001","article-title":"Generation and characterization of anti-\u03b1-enolase single-chain antibodies in chicken","volume":"137","author":"Leu","year":"2010","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1007\/s00253-006-0465-8","article-title":"Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems","volume":"72","author":"Terpe","year":"2006","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"20","DOI":"10.32607\/20758251-2009-1-3-20-28","article-title":"Phage display on the base of filamentous bacteriophages: Application for recombinant antibodies selection","volume":"1","author":"Tikunova","year":"2009","journal-title":"Acta Nat."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1007\/978-1-4939-7546-4_8","article-title":"The No-Nonsens SDS-PAGE","volume":"1721","author":"Backman","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1016\/0019-2791(71)90454-X","article-title":"Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G","volume":"8","author":"Engvall","year":"1971","journal-title":"Immunochemistry"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/8\/4146\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:48:58Z","timestamp":1760161738000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/8\/4146"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,16]]},"references-count":44,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,4]]}},"alternative-id":["ijms22084146"],"URL":"https:\/\/doi.org\/10.3390\/ijms22084146","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2021,4,16]]}}}