{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:47:19Z","timestamp":1760060839207,"version":"build-2065373602"},"reference-count":48,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T00:00:00Z","timestamp":1758758400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Agency for Research and Development (ANID)","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]},{"name":"ANID Millennium Science Initiative Program","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]},{"name":"ANID Doctoral Scholarship","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]},{"name":"FONDAP-ANID","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]},{"name":"POSDOCTORADO ANID","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]},{"name":"Bio &amp; Medical Technology Development Program of the National Research Foundation (NRF)","award":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"],"award-info":[{"award-number":["13220075","1241452","ICN17-022","21241768","1523A0007","3220635","RS-2025-15373195"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The development of effective tools to combat viral diseases remains a major challenge for the aquaculture industry. Infectious pancreatic necrosis virus (IPNv) is one of the most devastating pathogens affecting salmonids, leading to high mortality rates and substantial economic losses worldwide. Here, we present a novel nanobody discovery pipeline based on a Type IIS restriction enzyme-driven library assembly method that enables the rapid generation of highly diverse nanobody repertoires. This streamlined approach not only shortens the time required for nanobody identification but also offers remarkable adaptability, allowing its application to virtually any protein target, including antigens from aquaculture pathogens and beyond. By integrating this strategy with density gradient\u2013based enrichment and high-throughput screening, we successfully identified and validated a nanobody against the VP2 protein of IPNv, a key structural component essential for viral infectivity. These findings highlight the potential of this platform both as a versatile methodological advance in antibody engineering and as a practical foundation for developing innovative diagnostic and therapeutic tools. Ultimately, nanobodies generated through this pipeline could play a pivotal role in improving disease management and enhancing sustainability in aquaculture.<\/jats:p>","DOI":"10.3390\/ijms26199350","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T08:56:44Z","timestamp":1758790604000},"page":"9350","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Novel Type IIS-Based Library Assembly Technique for Developing Nanobodies Targeting IPNv VP2 Protein"],"prefix":"10.3390","volume":"26","author":[{"given":"Camila","family":"Pino-Belmar","sequence":"first","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johanna","family":"Himelreichs","sequence":"additional","affiliation":[{"name":"Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Camila","family":"Deride","sequence":"additional","affiliation":[{"name":"Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0486-985X","authenticated-orcid":false,"given":"Tamara","family":"Matute","sequence":"additional","affiliation":[{"name":"Agencia Nacional de Investigaci\u00f3n y Desarrollo\u2014Millennium Science Initiative Program\u2014Millennium Institute for Integrative Biology (iBio), Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"},{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4439-3745","authenticated-orcid":false,"given":"Isaac","family":"Nu\u00f1ez","sequence":"additional","affiliation":[{"name":"Agencia Nacional de Investigaci\u00f3n y Desarrollo\u2014Millennium Science Initiative Program\u2014Millennium Institute for Integrative Biology (iBio), Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"},{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Severine","family":"Cazaux","sequence":"additional","affiliation":[{"name":"Agencia Nacional de Investigaci\u00f3n y Desarrollo\u2014Millennium Science Initiative Program\u2014Millennium Institute for Integrative Biology (iBio), Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"},{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernan","family":"Federici","sequence":"additional","affiliation":[{"name":"Agencia Nacional de Investigaci\u00f3n y Desarrollo\u2014Millennium Science Initiative Program\u2014Millennium Institute for Integrative Biology (iBio), Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"},{"name":"Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Cat\u00f3lica de Chile, Santiago 8331150, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karen","family":"Moreno-Mendieta","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Genaro","family":"Soto-Rauch","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joaqu\u00edn","family":"Castro","sequence":"additional","affiliation":[{"name":"Bain & Company, Santiago 7550268, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Frenkel","sequence":"additional","affiliation":[{"name":"Bain & Company, Santiago 7550268, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joi-Hui","family":"Ho","sequence":"additional","affiliation":[{"name":"Bain & Company, Santiago 7550268, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Ascencios","sequence":"additional","affiliation":[{"name":"Bain & Company, Santiago 7550268, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7159-4146","authenticated-orcid":false,"given":"Daniel","family":"Sanhueza Teneo","sequence":"additional","affiliation":[{"name":"Institute of Immunology and Parasitology, Faculty of Medicine, Universidad Austral de Chile, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9","family":"Munizaga","sequence":"additional","affiliation":[{"name":"Biochemistry School, Faculty of Sciences, Universidad Austral de Chile, Valdivia 5110566, Chile"},{"name":"Facultad de Ciencias para el Cuidado de la Salud, Universidad San Sebasti\u00e1n, Valdivia 5090000, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Denise","family":"Haussmann","sequence":"additional","affiliation":[{"name":"Departamento de Ciencias B\u00e1sicas, Facultad de Ciencias, Universidad Santo Tom\u00e1s, Valdivia 5110566, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alejandro","family":"Rojas-Fernandez","sequence":"additional","affiliation":[{"name":"Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Valdivia 5110566, Chile"},{"name":"Berking Biotechnology, Valdivia 5090000, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaime Figueroa","family":"Valverde","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia 5110566, Chile"},{"name":"Interdisciplinary Center for Aquaculture Research (INCAR), Concepci\u00f3n 4030000, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guillermo","family":"Valenzuela-Nieto","sequence":"additional","affiliation":[{"name":"Facultad de Ciencias para el Cuidado de la Salud, Universidad San Sebasti\u00e1n, Valdivia 5090000, Chile"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/0959-8030(95)00003-8","article-title":"The molecular biology of infectious pancreatic necrosis virus (IPNV)","volume":"5","author":"Dobos","year":"1995","journal-title":"Annu. Rev. Fish Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/0042-6822(87)90180-2","article-title":"Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus","volume":"161","author":"Azad","year":"1987","journal-title":"Virology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1099\/0022-1317-71-2-299","article-title":"Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: A comparison with other Birnaviridae","volume":"71","author":"Kalland","year":"1990","journal-title":"J. Gen. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1016\/j.cell.2005.01.009","article-title":"The birnavirus crystal structure reveals structural relationships among icosahedral viruses","volume":"120","author":"Coulibaly","year":"2005","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1792","DOI":"10.1128\/JVI.01536-09","article-title":"Crystal structure of an Aquabirnavirus particle: Insights into antigenic diversity and virulence determinism","volume":"84","author":"Coulibaly","year":"2010","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2193","DOI":"10.1099\/0022-1317-67-10-2193","article-title":"Monoclonal antibodies to infectious pancreatic necrosis virus: Analysis of viral epitopes and comparison of different isolates","volume":"67","author":"Reno","year":"1986","journal-title":"J. Gen. Virol."},{"doi-asserted-by":"crossref","unstructured":"Asche, F., and Bj\u00f8rndal, T. (2011). The Economics of Salmon Aquaculture, Wiley.","key":"ref_7","DOI":"10.1002\/9781119993384"},{"doi-asserted-by":"crossref","unstructured":"Roberts, R.J. (2012). The virology of teleosts. Fish Pathology, Wiley-Blackwell.","key":"ref_8","DOI":"10.1002\/9781118222942"},{"key":"ref_9","first-page":"203","article-title":"Detecci\u00f3n del virus de la necrosis pancre\u00e1tica infecciosa (IPNV) en sedimentos de agua dulce","volume":"40","author":"Espinoza","year":"2008","journal-title":"Arch. Med. Vet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"108947","DOI":"10.1016\/j.fsi.2023.108947","article-title":"Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV)","volume":"140","author":"Sandoval","year":"2023","journal-title":"Fish Shellfish. Immunol."},{"doi-asserted-by":"crossref","unstructured":"Godoy, M., Kibenge, M.J.T., Montes de Oca, M., Pontigo, J.P., Coca, Y., Caro, D., Kusch, K., Suarez, R., Burbulis, I., and Kibenge, F.S.B. (2022). Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (Salmo salar) during 2021\u20132022. Pathogens, 11.","key":"ref_11","DOI":"10.3390\/pathogens11111368"},{"doi-asserted-by":"crossref","unstructured":"Hillestad, B., Johannessen, S., Melingen, G.O., and Moghadam, H.K. (2021). Identification of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant in Atlantic Salmon (Salmo salar L.) that can Cause High Mortality Even in Genetically Resistant Fish. Front. Genet., 12.","key":"ref_12","DOI":"10.3389\/fgene.2021.635185"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/s40259-019-00392-z","article-title":"The therapeutic potential of nanobodies","volume":"34","author":"Muyldermans","year":"2020","journal-title":"BioDrugs"},{"doi-asserted-by":"crossref","unstructured":"Wang, J., Kang, G., Yuan, H., Cao, X., Huang, H., and de Marco, A. (2021). Research progress and applications of multivalent, multispecific and modified nanobodies for disease treatment. Front. Immunol., 12.","key":"ref_14","DOI":"10.3389\/fimmu.2021.838082"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1038\/nrm4014","article-title":"Bricks and blueprints: Methods and standards for DNA assembly","volume":"16","author":"Casini","year":"2015","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1021\/sb4001504","article-title":"A golden gate modular cloning toolbox for plants","volume":"3","author":"Engler","year":"2014","journal-title":"ACS Synth. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1618","DOI":"10.1104\/pp.113.217661","article-title":"GoldenBraid 2.0: A comprehensive DNA assembly framework for plant synthetic biology","volume":"162","author":"Castelijns","year":"2013","journal-title":"Plant Physiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1039\/c0ib00070a","article-title":"DNA assembly for synthetic biology: From parts to pathways and beyond","volume":"3","author":"Ellis","year":"2011","journal-title":"Integr. Biol."},{"doi-asserted-by":"crossref","unstructured":"Engler, C., Kandzia, R., and Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. PLoS ONE, 3.","key":"ref_19","DOI":"10.1371\/journal.pone.0003647"},{"doi-asserted-by":"crossref","unstructured":"Andreou, A.I., and Nakayama, N. (2018). Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly. PLoS ONE, 13.","key":"ref_20","DOI":"10.1371\/journal.pone.0189892"},{"doi-asserted-by":"crossref","unstructured":"Pollak, B., Matute, T., Nu\u00f1ez, I., Cerda, A., Lopez, C., Vargas, V., Kan, A., Bielinski, V., von Dassow, P., and Dupont, C.L. (2020). Universal loop assembly: Open, efficient and cross-kingdom DNA fabrication. Synth. Biol., 5.","key":"ref_21","DOI":"10.1093\/synbio\/ysaa001"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1477","DOI":"10.1021\/acssynbio.8b00060","article-title":"Single Day Construction of Multigene Circuits with 3G Assembly","volume":"7","author":"Halleran","year":"2018","journal-title":"ACS Synth. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1021\/acssynbio.5b00124","article-title":"CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology","volume":"5","author":"Iverson","year":"2016","journal-title":"ACS Synth. Biol."},{"key":"ref_24","first-page":"e113","article-title":"MetClo: Methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme","volume":"46","author":"Lin","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.febslet.2013.10.044","article-title":"Production of prone-to-aggregate proteins","volume":"588","author":"Lebendiker","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.jbiotec.2008.05.007","article-title":"Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli","volume":"135","author":"Zou","year":"2008","journal-title":"J. Biotechnol."},{"doi-asserted-by":"crossref","unstructured":"Valenzuela Nieto, G., Jara, R., Watterson, D., Modhiran, N., Amarilla, A.A., Himelreichs, J., Khromykh, A.A., Salinas-Rebolledo, C., Pinto, T., and Cheuquemilla, Y. (2021). Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody. Sci. Rep., 11.","key":"ref_27","DOI":"10.1038\/s41598-021-82833-w"},{"doi-asserted-by":"crossref","unstructured":"Salema, V., Mar\u00edn, E., Mart\u00ednez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodel\u00f3n, G., and Fern\u00e1ndez, L.\u00c1. (2013). Selection of single domain antibodies from immune libraries displayed on the surface of, E. coli cells with two \u03b2-domains of opposite topologies. PLoS ONE, 8.","key":"ref_28","DOI":"10.1371\/journal.pone.0075126"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"W503","DOI":"10.1093\/nar\/gkn316","article-title":"IMGT\/V-QUEST: The highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis","volume":"36","author":"Brochet","year":"2008","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"252","DOI":"10.3390\/v7010252","article-title":"A polyprotein-expressing salmonid alphavirus replicon induces modest protection in atlantic salmon (Salmo salar) against infectious pancreatic necrosis","volume":"7","author":"Abdullah","year":"2015","journal-title":"Viruses"},{"doi-asserted-by":"crossref","unstructured":"Levican, J., Miranda-C\u00e1rdenas, C., Soto-Rifo, R., Aguayo, F., Gaggero, A., and Le\u00f3n, O. (2017). Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis. Sci. Rep., 7.","key":"ref_31","DOI":"10.1038\/s41598-017-03036-w"},{"doi-asserted-by":"crossref","unstructured":"Isaacs, A., Nieto, G.V., Zhang, X., Modhiran, N., Barr, J., Thakur, N., Low, Y.S., Parry, R.H., Barnes, J.B., and Jara, R. (2025). A nanobody-based therapeutic targeting Nipah virus limits viral escape. Nat. Struct. Mol. Biol.","key":"ref_32","DOI":"10.1038\/s41594-025-01598-2"},{"doi-asserted-by":"crossref","unstructured":"Valenzuela-Nieto, G., Miranda-Chacon, Z., Salinas-Rebolledo, C., Jara, R., Cuevas, A., Berking, A., and Rojas-Fernandez, A. (2022). Nanobodies: COVID-19 and Future Perspectives. Front. Drug Discov., 2.","key":"ref_33","DOI":"10.3389\/fddsv.2022.927164"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"107085","DOI":"10.1016\/j.isci.2023.107085","article-title":"A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants","volume":"26","author":"Modhiran","year":"2023","journal-title":"Iscience"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"109869","DOI":"10.1016\/j.celrep.2021.109869","article-title":"A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration","volume":"37","author":"Wu","year":"2021","journal-title":"Cell Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4635","DOI":"10.1038\/s41467-021-24905-z","article-title":"A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection","volume":"12","author":"Li","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4420","DOI":"10.1038\/s41467-020-18174-5","article-title":"An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction","volume":"11","author":"Hanke","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"6500","DOI":"10.2174\/1381612822666160923114417","article-title":"Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies","volume":"22","author":"Romao","year":"2016","journal-title":"Curr. Pharm. Des."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1586\/14760584.4.1.89","article-title":"Vaccines for fish in aquaculture","volume":"4","author":"Sommerset","year":"2005","journal-title":"Expert Rev. Vaccines"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.2217\/nnm.13.86","article-title":"Nanobodies and their potential applications","volume":"8","author":"Devoogdt","year":"2013","journal-title":"Nanomedicine"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.jbiotec.2016.07.025","article-title":"Characterization of nanobodies binding human fibrinogen selected by, E. coli display","volume":"234","author":"Salema","year":"2016","journal-title":"J. Biotechnol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"7346","DOI":"10.1074\/jbc.M007734200","article-title":"Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs","volume":"276","author":"Lauwereys","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"doi-asserted-by":"crossref","unstructured":"Wohlwend, J., Corso, G., Passaro, S., Getz, N., Reveiz, M., Leidal, K., Swiderski, W., Atkinson, L., Portnoi, T., and Chinn, I. (2025). Boltz-1 Democratizing Biomolecular Interaction Modeling. BioRxiv.","key":"ref_44","DOI":"10.1101\/2024.11.19.624167"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.1038\/s41467-022-28865-w","article-title":"Improved prediction of protein-protein interactions using AlphaFold2","volume":"13","author":"Bryant","year":"2022","journal-title":"Nat. Commun."},{"doi-asserted-by":"crossref","unstructured":"Basu, S., and Wallner, B. (2016). DockQ: A Quality Measure for Protein-Protein Docking Models. PLoS ONE, 11.","key":"ref_47","DOI":"10.1371\/journal.pone.0161879"},{"doi-asserted-by":"crossref","unstructured":"Papadopoulos, A.-M., Axenopoulos, A., Iatrou, A., Stamatopoulos, K., Alvarez, F., and Daras, P. (2025). ParaSurf: A surface-based deep learning approach for paratope-antigen interaction prediction. Bioinformatics, 41.","key":"ref_48","DOI":"10.1093\/bioinformatics\/btaf062"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9350\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:49:18Z","timestamp":1760035758000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9350"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,25]]},"references-count":48,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms26199350"],"URL":"https:\/\/doi.org\/10.3390\/ijms26199350","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,9,25]]}}}