{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T02:32:57Z","timestamp":1768530777251,"version":"3.49.0"},"reference-count":50,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T00:00:00Z","timestamp":1737676800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH-NINDS","doi-asserted-by":"publisher","award":["R15NS128563"],"award-info":[{"award-number":["R15NS128563"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Snakebite is a critical global public health issue, causing substantial mortality and morbidity, particularly in tropical and subtropical regions. The development of innovative antivenoms targeting snake venom toxins is therefore of paramount importance. In this study, we adopted an epitope-directed approach to design three degenerate 15-mer peptides based on amino acid sequence alignments of snake venom phospholipase A2s (PLA2s) and snake venom serine proteases (SVSPs) from snake (Crotalus atrox). By leveraging their immunogenic and inhibitory profiles, these peptides were specifically designed to target the Asp49 and Lys49 variants of PLA2 and SVSP toxins. Groups of five mice were immunized with each peptide, and IgG mRNA was subsequently extracted from peripheral blood mononuclear cells (PBMCs) and spleen lymphocytes of the top three responders. The extracted mRNA was reverse-transcribed into complementary DNA (cDNA), and the variable regions of the IgG heavy and kappa chains were amplified using polymerase chain reaction (PCR). These amplified regions were then linked with a 66-nucleotide spacer to construct single-chain variable fragments (scFvs). Sequence analysis of 48 randomly selected plasmids from each PLA2 and SVSP scFv library revealed that over 80% contained scFv sequences with notable diversity observed in the complementarity-determining regions (CDRs), particularly CDR3. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the SP peptide elicited a broader immune response in mice compared to the Asp49 peptide, implying the strong immunogenicity of the SP peptide. These scFvs represent a promising foundation for the development of recombinant human monoclonal antibodies targeting snake PLA2 and SVSP toxins, providing a potential therapeutic strategy for the treatment of snakebites.<\/jats:p>","DOI":"10.3390\/toxins17020055","type":"journal-article","created":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T09:40:39Z","timestamp":1737711639000},"page":"55","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Single-Chain Variable Fragments: Targeting Snake Venom Phospholipase A2 and Serine Protease"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0740-8028","authenticated-orcid":false,"given":"Ying","family":"Jia","sequence":"first","affiliation":[{"name":"School of Integrative Biological and Chemical Sciences, University of Texas Rio Grande Valley, Brownsville, TX 78520, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ariane","family":"Garcia","sequence":"additional","affiliation":[{"name":"School of Integrative Biological and Chemical Sciences, University of Texas Rio Grande Valley, Brownsville, TX 78520, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth","family":"Reyes","sequence":"additional","affiliation":[{"name":"School of Integrative Biological and Chemical Sciences, University of Texas Rio Grande Valley, Brownsville, TX 78520, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1186\/s40409-017-0127-6","article-title":"Snakebite envenomation turns again into a neglected tropical disease!","volume":"23","author":"Chippaux","year":"2017","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"17063","DOI":"10.1038\/nrdp.2017.63","article-title":"Snakebite envenoming","volume":"3","author":"Calvete","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.toxicon.2019.05.001","article-title":"Comparative Venomics of Brazilian Coral Snakes: Micrurus frontalis, Micrurus spixii spixii, and Micrurus surinamensis","volume":"166","author":"Sanz","year":"2019","journal-title":"Toxicon"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100070","DOI":"10.1016\/j.toxcx.2021.100070","article-title":"Mutual enlightenment: A toolbox of concepts and methods for integrating evolutionary and clinical toxinology via snake venomics and contextual stance","volume":"9\u201310","author":"Calvete","year":"2021","journal-title":"Toxicon X"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Sunagar, K., Khochare, S., Senji Laxme, R.R., Attarde, S., Dam, P., Suranse, V., Khaire, A., Martin, G., and Captain, A. (2021). A wolf in another wolf\u2019s clothing: Post-genomic regulation dictates venom profiles of medically-important cryptic kraits in India. Toxins, 13.","DOI":"10.7934\/P3897"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Tasoulis, T., and Isbister, G.K. (2017). A review and database of snake venom proteomes. Toxins, 9.","DOI":"10.3390\/toxins9090290"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2085536","DOI":"10.1080\/19420862.2022.2085536","article-title":"In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom","volume":"14","author":"Ledsgaard","year":"2022","journal-title":"Mabs"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1038\/s41467-023-36393-4","article-title":"Discovery and optimization of a broadly- neutralizing human monoclonal antibody against long-chain \u03b1-neurotoxins from snakes","volume":"14","author":"Ledsgaard","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1126\/scitranslmed.adk1867","article-title":"Synthetic development of a broadly neutralizing antibody against snake venom long-chain \u03b1-neurotoxins","volume":"16","author":"Khalek","year":"2024","journal-title":"Sci. Transl. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"193","DOI":"10.3390\/antib2020193","article-title":"In vivo applications of single chain Fv (variable domain) (scFv) fragments","volume":"2","author":"Monnier","year":"2013","journal-title":"Antibodies"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sandomenico, A., Sivaccumar, J.P., and Ruvo, M. (2020). Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176324"},{"key":"ref_12","first-page":"351","article-title":"Construction and characterization of minibodies for imaging and therapy of colorectal carcinomas","volume":"207","author":"Yazaki","year":"2003","journal-title":"Methods Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3884","DOI":"10.1200\/JCO.2012.42.4887","article-title":"Advances in immuno-positron emission tomography: Antibodies for molecular imaging in oncology","volume":"30","author":"Knowles","year":"2012","journal-title":"J. Clin. Oncol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1126","DOI":"10.1038\/nbt1142","article-title":"Engineered antibody fragments and the rise of single domains","volume":"23","author":"Holliger","year":"2005","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/nbt0409-331","article-title":"Development trends for therapeutic antibody fragments","volume":"27","author":"Nelson","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_16","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_17","doi-asserted-by":"crossref","first-page":"980250","DOI":"10.1155\/2012\/980250","article-title":"scFv antibody: Principles and clinical application","volume":"2012","author":"Ahmad","year":"2012","journal-title":"Clin. Dev. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Bates, A., and Power, C.A. (2019). David vs. Goliath: The structure, function, and clinical prospects of antibody fragments. Antibodies, 8.","DOI":"10.3390\/antib8020028"},{"key":"ref_19","unstructured":"Campbell, J.A., and Lamar, W.W. (2004). The Venomous Reptiles of the Western Hemisphere, Comstock Publishing Associates."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3055","DOI":"10.1021\/pr900249q","article-title":"Exploring the venom proteome of the western diamondback rattlesnake, Crotalus atrox, via snake venomics and combinatorial peptide ligand library approaches","volume":"8","author":"Calvete","year":"2009","journal-title":"J. Proteome Res."},{"key":"ref_21","first-page":"18","article-title":"Toxin transcripts in Crotalus atrox venom and in silico structure of toxins","volume":"10","author":"Jia","year":"2020","journal-title":"J. Venom Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"14402","DOI":"10.1016\/S0021-9258(18)47808-8","article-title":"Structure-function relationships of phospholipases","volume":"262","author":"Kini","year":"1987","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1093\/oxfordjournals.jbchem.a003101","article-title":"Phospholipase A2","volume":"131","author":"Murakami","year":"2002","journal-title":"J. Biochem"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2","DOI":"10.33425\/2689-1050.1060","article-title":"Tarantula toxin transcripts and toxin structure prediction by AlphaFold","volume":"6","author":"Vega","year":"2024","journal-title":"Chem. Pharm. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.toxicon.2012.09.003","article-title":"The long road of research on snake venom serine proteinases","volume":"62","author":"Serrano","year":"2013","journal-title":"Toxicon"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.1110\/ps.035436.108","article-title":"Unconventional serine proteinases: Variations on the catalytic ser\/His\/Asp triad configuration","volume":"17","author":"Ekici","year":"2008","journal-title":"Protein Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1126\/science.6369538","article-title":"Evolution of proteolytic enzymes","volume":"224","author":"Neurath","year":"1984","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.1016\/j.toxicon.2005.02.016","article-title":"Practical application of snake venom toxins in haemostasis","volume":"45","author":"Marsh","year":"2005","journal-title":"Toxicon"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.toxicon.2017.10.022","article-title":"Identification of B cell recognized linear epitopes in a snake venom serine proteinase from the central American bushmaster Lachesis stenophrys","volume":"140","author":"Madrigal","year":"2017","journal-title":"Toxicon"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e4497","DOI":"10.1002\/pro.4497","article-title":"BepiPred-3.0: Improved B-cell epitope prediction using protein language models","volume":"31","author":"Clifford","year":"2022","journal-title":"Protein Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1016\/j.toxicon.2012.05.028","article-title":"Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A2 homologue from Bothrops asper snake venom","volume":"60","author":"Lomonte","year":"2012","journal-title":"Toxicon"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1016\/j.bbamem.2007.01.023","article-title":"Mapping of the structural determinants of artificial and biological membrane damaging activities of Lys49 phospholipase A2 by scanning alanine mutagenesis","volume":"1768","author":"Chioato","year":"2007","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_33","first-page":"2265","article-title":"A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms","volume":"12","author":"Fernandes","year":"2014","journal-title":"Biochem. Biophys. Acta"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"de Castro, K.L.P., Lopes-de-Souza, L., de Oliveira, D., Machado-de-\u00c1vila, R.A., Paiva, A.L.B., de Freitas, C.F., Ho, P.L., Ch\u00e1vez-Ol\u00f3rtegui, C., and Guerra, C. (2019). A combined strategy to improve the development of a coral antivenom against Micrurus spp.. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.02422"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.toxicon.2014.10.015","article-title":"Identification and characterization of B-cell epitopes of 3FTx and PLA2 toxins from Micrurus corallinus snake venom","volume":"93","author":"Castro","year":"2015","journal-title":"Toxicon"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4544","DOI":"10.1111\/j.1742-4658.2011.08115.x","article-title":"Enzymatic toxins from snake venom: Structural characterization and mechanism of catalysis","volume":"278","author":"Kang","year":"2011","journal-title":"FEBS J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1023\/A:1008975123362","article-title":"Molecular farming of pharmaceutical proteins","volume":"9","author":"Fischer","year":"2000","journal-title":"Transgenic Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3393","DOI":"10.1182\/blood-2006-07-030593","article-title":"Cell-free production of scFv fusion proteins: An efficient approach for personalized lymphoma vaccines","volume":"109","author":"Kanter","year":"2007","journal-title":"Blood"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Chung, D.H., Kong, S., Young, N.J., Chuo, S.-W., Shiah, J.V., Connelly, E.J., Rohweder, P.J., Born, A., Manglik, A., and Grandis, J.R. (2023). Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets. Commun. Biol., 6.","DOI":"10.1038\/s42003-023-05390-0"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Keyser, P.D., Kalichuk, V., Z\u00f6gg, T., Wohlk\u00f6nig, A., Schenck, S., Brunner, J., Pardon, E., and Steyaert, J. (2024). A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery. Biosens. Bioelectron., 271.","DOI":"10.1016\/j.bios.2024.116951"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2151","DOI":"10.1016\/j.drudis.2022.05.002","article-title":"Advances in antibody phage display technology","volume":"27","author":"Ledsgaad","year":"2022","journal-title":"Drug Discov. Today"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Palma, M. (2023). Epitopes and mimotopes identification using phage display for vaccine development against infectious pathogens. Vaccines, 11.","DOI":"10.3390\/vaccines11071176"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1016\/0161-5890(87)90058-7","article-title":"Regulation of kappa\/lambda immunoglobulin light chain expression in normal murine lymphocytes","volume":"24","author":"Woloschak","year":"1987","journal-title":"Mol. Immunol."},{"key":"ref_44","unstructured":"Kabat, E.A., Wu, T., Gottesman, K.S., Perry, H.M., and Foeller, C. (1991). Sequences of Proteins of Immunological Interest, Diane Publishing Co.. [5th ed.]."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1016\/0959-440X(94)90267-4","article-title":"Antibody-antigen interactions: New structures and new conformational changes","volume":"4","author":"Wilson","year":"1994","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1101\/gr.849004","article-title":"WebLogo: A sequence logo generator","volume":"14","author":"Crooks","year":"2004","journal-title":"Genome Res."},{"key":"ref_47","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"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1002\/jmr.2178","article-title":"Indirect ELISA-based approach for comparative measurement of high-affinity cohesin-dockerin interactions","volume":"25","author":"Slutzki","year":"2012","journal-title":"J. Mol. Recognit."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4967","DOI":"10.1128\/AEM.00435-12","article-title":"Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin","volume":"78","author":"Wang","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_50","first-page":"25","article-title":"Construction and screening of a phage display library of single chain Fv antibody efficiently from mouse immunized with ovalbumin","volume":"38","author":"Lang","year":"2018","journal-title":"China Biotechnol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/17\/2\/55\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T10:35:43Z","timestamp":1759919743000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/17\/2\/55"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,24]]},"references-count":50,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,2]]}},"alternative-id":["toxins17020055"],"URL":"https:\/\/doi.org\/10.3390\/toxins17020055","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,24]]}}}